Hyaluronic acid-binding synthetic peptidoglycans, preparation, and methods of use

ABSTRACT

This invention pertains to the field of hyaluronic acid-binding synthetic peptidoglycans and methods of forming and using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/489,602 filed May 24, 2011 and U.S.Provisional Application Ser. No. 61/550,621 filed Oct. 24, 2011. Thedisclosures of both of which are incorporated herein by reference.

TECHNICAL FIELD

This invention pertains to the field of hyaluronic acid-bindingsynthetic peptidoglycans and methods of forming and using the same.

BACKGROUND AND SUMMARY OF THE INVENTION

Articular cartilage is an important component for the protection ofbones in the body. In particular, articular cartilage functions toprotect articulating bones from damage by providing a near-frictionlesssurface for bone movement and also providing compressive strength tojoints. Articular cartilage broadly includes an extracellular matrix(ECM) derived from three main components: a collagen scaffold,hyaluronic acid (HA), and aggrecan. The material composition ofarticular cartilage dictates the tissue's biological, chemical andmechanical properties. The extracellular matrix (ECM) of healthycartilage is primarily composed of a network of collagen fibrils (15-22%wet weight type II collagen), proteoglycans (4-7% wet weight),glycoproteins, water (60-85%) and electrolytes, giving rise to aviscoelastic tissue with depth-dependent structural and mechanicalanisotropy.

Cartilage degradation and wear is a hallmark of osteoarthritis (OA).During the initial stages of OA, aggrecan, a major proteoglycan incartilage, is an early component to be degraded. The aggrecan monomer isa protein core with covalently attached glycosaminoglycan (GAG) sidechains that bind to filamentous hyaluronic acid via globular domains anda link protein. Proteases, such as aggrecanases, cleave aggrecan atspecific sites creating protein fragments and free GAG chains that areunable to rebind to HA. Instead, these free GAG chains are extruded fromthe matrix, which not only reduces compressive strength, but alsoinitiates an increase in pro-inflammatory cytokines and matrixmetalloproteases. The presence of aggrecan has been shown to reducediffusion of proteases protecting underlying collagen fibers fromproteolytic cleavage. Loss of aggrecan occurs even in normal cartilageand is not immediately correlated to OA. However, loss of type IIcollagen is considered an irreversible process, leading to precociouswear.

Osteoarthritis is the most common form of arthritis, affecting 27million people in the US alone. The most prevalent symptoms ofosteoarthritis include immense pain, stiffening in the joints, andtender and inflamed joints. Advanced stages of osteoarthritis can leadto complete degradation of the articular cartilage, causing immobilityof joints and damage to the underlying bone. The direct costs ofarthritis in the United States are estimated to be approximately $185.5billion each year.

Although lifestyle changes and multiple medications are often used forthe treatment of osteoarthritis, there has been little success inregeneration of defective cartilage and relieving the symptoms caused bythe loss of cartilage. This inability to halt the progression ofosteoarthritis and repair the existing damage typically leads to aninvasive, end stage cartilage replacement procedure. Thus, analternative treatment option for osteoarthritis is highly desired.

The present disclosure describes improved biomaterials for cartilageregeneration, including hyaluronic acid-binding synthetic peptidoglycansthat can be utilized to restore damaged cartilage in an affectedpatient, along with methods of forming and using the syntheticpeptidoglycans. Furthermore, the hyaluronic acid-binding syntheticpeptidoglycans are designed to functionally mimic aggrecan, resistaggrecanase degradation, and limit proteolytic degradation. The absenceof the native amino acid sequence seen in aggrecan makes these moleculesless susceptible to proteolytic cleavage.

The following numbered embodiments are contemplated and arenon-limiting:

1. A hyaluronic acid-binding synthetic peptidoglycan comprising asynthetic peptide conjugated to a glycan wherein the synthetic peptidecomprises a hyaluronic acid-binding sequence.

2. The synthetic peptidoglycan of clause 1 wherein the synthetic peptidecomprises an amino acid sequence of the formulaB1-X1-X2-X3-X4-X5-X6-X7-X8-B2,

-   -   wherein X8 is present or is not present,    -   wherein B1 is a basic amino acid,    -   wherein B2 is a basic amino acid, and    -   wherein X1-X8 are non-acidic amino acids.

3. The synthetic peptidoglycan of clause 1 or clause 2 wherein thesynthetic peptide comprises an amino acid sequence selected from thegroup consisting of:

GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK;SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP;SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR;RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR;RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK;RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK;  and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have aglycine-cysteine (GC) attached to the C-terminus of the peptide, or aglycine-cysteine-glycine (GCG) attached to the N-terminus.

4. The synthetic peptidoglycan of any one of clauses 1 to 3 wherein theglycan is selected from the group consisting of dextran, chondroitin,chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin,keratin, keratan sulfate, and hyaluronic acid.

5. The synthetic peptidoglycan of any one of clauses 1 to 4 wherein theglycan is selected from the group consisting of chondroitin sulfate andkeratan sulfate.

6. The synthetic peptidoglycan of any one of clauses 1 to 5 wherein thesynthetic peptidoglycan is resistant to aggrecanase.

7. The synthetic peptidoglycan of any one of clauses 1 to 6 wherein thesynthetic peptidoglycan is lyophilized.

8. A compound of formula P_(n)G_(x) wherein n is 1 to 20;

wherein x is 1 to 20;

wherein P is a synthetic peptide of about 5 to about 40 amino acidscomprising a hyaluronic acid binding sequence; and

wherein G is a glycan. 9. A compound of formula (P_(n)L)_(x)G wherein nis 1 to 20;

wherein x is 1 to 20;

wherein P is a synthetic peptide of about 5 to about 40 amino acidscomprising a hyaluronic acid binding sequence;

wherein L is a linker; and

wherein G is a glycan. 10. A compound of formula P(LG_(n))_(x) wherein nis 1 to 20;

wherein x is 1 to 20;

wherein P is a synthetic peptide of about 5 to about 40 amino acidscomprising a hyaluronic acid binding sequence;

wherein L is a linker; and

wherein G is a glycan.

11. A compound of formula P_(n)G_(x) wherein n is MWG/1000;

wherein MWG is the molecular weight of G rounded to the nearest 1 kDa;

wherein x is 1 to 20;

wherein P is a synthetic peptide of about 5 to about 40 amino acidscomprising a hyaluronic acid binding sequence; and

wherein G is a glycan.

12. A compound of formula (P_(n)L)_(x)G wherein n is MWG/1000;

wherein MWG is the molecular weight of G rounded to the nearest 1 kDa;

wherein x is 1 to 20;

wherein P is a synthetic peptide of about 5 to about 40 amino acidscomprising a hyaluronic acid binding sequence;

wherein L is a linker; and

wherein G is a glycan.

13. The compound of any one of clauses 8 to 12 wherein the syntheticpeptide comprises an amino acid sequence of the formulaB1-X1-X2-X3-X4-X5-X6-X7-X8-B2,

wherein X8 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid, and

wherein X1-X8 are non-acidic amino acids.

14. The compound of any one of clauses 8 to 13 wherein the syntheticpeptide comprises an amino acid sequence selected from the groupconsisting of:

GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK;SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP;SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR;RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR;RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK;RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK;  and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have aglycine-cysteine (GC) attached to the C-terminus of the peptide, or aglycine-cysteine-glycine (GCG) attached to the N-terminus.

15. The compound of any one of clauses 8 to 14 wherein the glycan isselected from the group consisting of dextran, chondroitin, chondroitinsulfate, dermatan, dermatan sulfate, heparan, heparin, keratin, keratansulfate, and hyaluronic acid.

16. The compound of any one of clauses 8 to 15 wherein the glycan isselected from the group consisting of chondroitin sulfate and keratansulfate.

17. The compound of any one of clauses 8 to 16 wherein the syntheticpeptidoglycan is resistant to aggrecanase.

18. An engineered collagen matrix comprising polymerized collagen,hyaluronic acid, and a hyaluronic acid-binding synthetic peptidoglycan.

19. The engineered collagen matrix of clause 18 wherein the collagen isselected from the group consisting of type I collagen, type II collagen,type III collagen, type IV collagen, type IX collagen, type XI collagen,and combinations thereof.

20. The engineered collagen matrix of clause 18 or 19 wherein thepeptide component of the synthetic peptidoglycan comprises an amino acidsequence of the formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2,

wherein X8 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid, and

wherein X1-X8 are non-acidic amino acids.

21. The engineered collagen matrix of any one of clauses 18 to 20wherein the peptide component of the synthetic peptidoglycan comprisesan amino acid sequence selected from the group consisting of:

GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK;SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP;SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR;RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR;RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK;RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK;  and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have aglycine-cysteine (GC) attached to the C-terminus of the peptide, or aglycine-cysteine-glycine (GCG) attached to the N-terminus.

22. The engineered collagen matrix of any one of clauses 18 to 21wherein the glycan component of the synthetic peptidoglycan is selectedfrom the group consisting of dextran, chondroitin, chondroitin sulfate,dermatan, dermatan sulfate, heparan, heparin, keratin, keratan sulfate,and hyaluronic acid.

23. The engineered collagen matrix of any one of clauses 18 to 22wherein the glycan component of the synthetic peptidoglycan is selectedfrom the group consisting of chondroitin sulfate and keratan sulfate.

24. The engineered collagen matrix of any one of clauses 18 to 23wherein the synthetic peptidoglycan is resistant to aggrecanase.

25. The engineered collagen matrix of any one of clauses 18 to 24wherein the matrix is effective as a tissue graft.

26. The engineered collagen matrix of clause 25 wherein the tissue graftis implanted into a patient.

27. The engineered collagen matrix of any one of clauses 18 to 24wherein the matrix is in the form of a gel.

28. The engineered collagen matrix of clause 27 wherein the gel isadministered to a patient by injection.

29. The engineered collagen matrix of any one of clauses 18 to 24wherein the matrix is effective as a composition for in vitro culture ofcells.

30. The engineered collagen matrix of clause 29 wherein the matrixfurther comprises an exogenous population of cells.

31. The engineered collagen matrix of clause 30 wherein the cells areselected from the group consisting of chondrocytes and stem cells.

32. The engineered collagen matrix of clause 31 wherein the stem cellsare selected from the group consisting of osteoblasts, osteogenic cells,and mesenchymal stem cells.

33. The engineered collagen matrix of any one of clauses 18 to 32further comprising one or more nutrients.

34. The engineered collagen matrix of any one of clauses 18 to 33further comprising one or more growth factors.

35. The engineered collagen matrix of any one of clauses 18 to 34wherein the matrix is sterilized.

36. A composition for in vitro culture of chondrocytes or stem cellscomprising a hyaluronic acid-binding synthetic peptidoglycan.

37. The composition of clause 36 wherein the peptide component of thesynthetic peptidoglycan comprises an amino acid sequence of the formulaB1-X1-X2-X3-X4-X5-X6-X7-X8-B2,

wherein X8 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid, and

wherein X1-X8 are non-acidic amino acids.

38. The composition of clause 36 or clause 37 wherein the peptidecomponent of the synthetic peptidoglycan comprises an amino acidsequence selected from the group consisting of:

GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK;SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP;SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR;RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR;RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK;RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK;  and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have aglycine-cysteine (GC) attached to the C-terminus of the peptide, or aglycine-cysteine-glycine (GCG) attached to the N-terminus.

39. The composition of any one of clauses 36 to 38 wherein the glycancomponent of the synthetic peptidoglycan is selected from the groupconsisting of dextran, chondroitin, chondroitin sulfate, dermatan,dermatan sulfate, heparan, heparin, keratin, keratan sulfate, andhyaluronic acid.

40. The composition of any one of clauses 36 to 39 wherein the glycancomponent of the synthetic peptidoglycan is selected from the groupconsisting of chondroitin sulfate and keratan sulfate.

41. The composition of any one of clauses 36 to 40 wherein the syntheticpeptidoglycan is resistant to aggrecanase.

42. The composition of any one of clauses 36 to 41 wherein the stemcells are selected from the group consisting of osteoblasts, osteogeniccells, and mesenchymal stem cells.

43. The composition of any one of clauses 36 to 42 further comprisingone or more nutrients.

44. The composition of any one of clauses 36 to 43 further comprisingone or more growth factors.

45. The composition of any one of clauses 36 to 44 wherein thecomposition is sterilized.

46. An additive for a biomaterial cartilage or bone replacementcomposition comprising a hyaluronic acid-binding synthetic peptidoglycanfor addition to an existing biomaterial cartilage or bone replacementmaterial.

47. The additive of clause 46 wherein the peptide component of thesynthetic peptidoglycan comprises an amino acid sequence of the formulaB1-X1-X2-X3-X4-X5-X6-X7-X8-B2,

wherein X8 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid, and

wherein X1-X8 are non-acidic amino acids.

48. The additive of clause 46 or clause 47 wherein the peptide componentof the synthetic peptidoglycan comprises an amino acid sequence selectedfrom the group consisting of:

GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK;SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP;SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR;RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR;RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK;RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK;  and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have aglycine-cysteine (GC) attached to the C-terminus of the peptide, or aglycine-cysteine-glycine (GCG) attached to the N-terminus.

49. The additive of any one of clauses 46 to 48 wherein the glycancomponent of the synthetic peptidoglycan is selected from the groupconsisting of dextran, chondroitin, chondroitin sulfate, dermatan,dermatan sulfate, heparan, heparin, keratin, keratan sulfate, andhyaluronic acid.

50. The additive of any one of clauses 46 to 49 wherein the glycan isselected from the group consisting of chondroitin sulfate and keratansulfate.

51. The additive of any one of clauses 46 to 50 wherein the syntheticpeptidoglycan is resistant to aggrecanase.

52. A method of treatment for arthritis in a patient, said methodcomprising the step of administering to the patient a hyaluronicacid-binding synthetic peptidoglycan, wherein the syntheticpeptidoglycan reduces a symptom associated with the arthritis.

53. The method of clause 52 wherein the peptide component of thesynthetic peptidoglycan comprises an amino acid sequence of the formulaB1-X1-X2-X3-X4-X5-X6-X7-X8-B2,

wherein X8 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid, and

wherein X1-X8 are non-acidic amino acids.

54. The method of clause 52 or clause 53 wherein the peptide componentof the synthetic peptidoglycan comprises an amino acid sequence selectedfrom the group consisting of:

GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK;SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP;SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR;RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR;RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK;RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK;  and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have aglycine-cysteine (GC) attached to the C-terminus of the peptide, or aglycine-cysteine-glycine (GCG) attached to the N-terminus.

55. The method of any one of clauses 52 to 54 wherein the glycancomponent of the synthetic peptidoglycan is selected from the groupconsisting of dextran, chondroitin, chondroitin sulfate, dermatan,dermatan sulfate, heparan, heparin, keratin, keratan sulfate, andhyaluronic acid.

56. The method of any one of clauses 52 to 55 wherein the glycan isselected from the group consisting of chondroitin sulfate and keratansulfate.

57. The method of any one of clauses 52 to 56 wherein the syntheticpeptidoglycan is resistant to aggrccanasc.

58. The method of any one of clauses 52 to 57 wherein the arthritis isosteoarthritis.

59. The method of any one of clauses 52 to 57 wherein the arthritis isrheumatoid arthritis.

60. The method of any one of clauses 52 to 59 wherein the syntheticpeptidoglycan is administered to the patient by injection.

61. The method of clause 60 wherein the injection is an intraarticularinjection.

62. The method of clause 60 wherein the injection is into a jointcapsule of the patient.

63. The method of any one of clauses 52 to 62 wherein the syntheticpeptidoglycan is administered using a needle or a device for infusion.

64. The method of any one of clauses 52 to 63 wherein the syntheticpeptidoglycan acts as a lubricant.

65. The method of any one of clauses 52 to 64 wherein the syntheticpeptidoglycan prevents bone on bone articulation or prevents cartilageloss.

66. A method of preparing a biomaterial or bone cartilage replacement,said method comprising the step of combining the synthetic peptidoglycanand an existing biomaterial or bone cartilage replacement material.

67. The method of clause 66 wherein the peptide component of thesynthetic peptidoglycan comprises an amino acid sequence of the formulaB1-X1-X2-X3-X4-X5-X6-X7-X8-B2,

wherein X8 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid, and

wherein X1-X8 are non-acidic amino acids.

68. The method of clause 66 or clause 67 wherein the peptide componentof the synthetic peptidoglycan comprises an amino acid sequence selectedfrom the group consisting of:

GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK;SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP;SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKTKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR;RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR;RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK;RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK;  and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have aglycine-cysteine (GC) attached to the C-terminus of the peptide, or aglycine-cysteine-glycine (GCG) attached to the N-terminus.

69. The method of any one of clauses 66 to 68 wherein the glycancomponent of the synthetic peptidoglycan is selected from the groupconsisting of dextran, chondroitin, chondroitin sulfate, dermatan,dermatan sulfate, heparan, heparin, keratin, keratan sulfate, andhyaluronic acid.

70. The method of any one of clauses 66 to 69 wherein the glycan isselected from the group consisting of chondroitin sulfate and keratansulfate.

71. The method of any one of clauses 66 to 70 wherein the syntheticpeptidoglycan is resistant to aggrecanase.

72. A method of reducing or preventing hyaluronic acid degradation in apatient, said method comprising administering to the patient ahyaluronic acid-binding synthetic peptidoglycan.

73. The method of clause 72 wherein the peptide component of thesynthetic peptidoglycan comprises an amino acid sequence of the formulaB1-X1-X2-X3-X4-X5-X6-X7-X8-B2,

wherein X8 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid, and

wherein X1-X8 are non-acidic amino acids.

74. The method of clause 72 or clause 73 wherein the peptide componentof the synthetic peptidoglycan comprises an amino acid sequence selectedfrom the group consisting of:

GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK;SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP;SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR;RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR;RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK;RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK;  and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have aglycine-cysteine (GC) attached to the C-terminus of the peptide, or aglycine-cysteine-glycine (GCG) attached to the N-terminus

75. The method of any one of clauses 72 to 74 wherein the glycancomponent of the synthetic peptidoglycan is selected from the groupconsisting of dextran, chondroitin, chondroitin sulfate, dermatan,dermatan sulfate, heparan, heparin, keratin, keratan sulfate, andhyaluronic acid.

76. The method of any one of clauses 72 to 75 wherein the glycan isselected from the group consisting of chondroitin sulfate and keratansulfate.

77. The method of any one of clauses 72 to 76 wherein the syntheticpeptidoglycan is resistant to aggrecanase.

78. The method of any one of clauses 72 to 77 wherein the syntheticpeptidoglycan is administered to the patient by injection.

79. The method of clause 78 wherein the injection is an intraarticularinjection.

80. The method of clause 78 wherein the injection is into a jointcapsule of the patient.

81. The method of any one of clauses 72 to 80 wherein the rate ofhyaluronic acid degradation is reduced.

82. A method for correcting or modifying a tissue defect in a patientcomprising

administering into the tissue defect a hyaluronic acid-binding syntheticpeptidoglycan wherein the defect is corrected or modified.

83. The method of clause 82 wherein the peptide component of thesynthetic peptidoglycan comprises an amino acid sequence of the formulaB1-X1-X2-X3-X4-X5-X6-X7-X8-B2,

wherein X8 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid, and

wherein X1-X8 are non-acidic amino acids.

84. The method of clause 82 or clause 83 wherein the peptide componentof the synthetic peptidoglycan comprises an amino acid sequence selectedfrom the group consisting of:

GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK;SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP;SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR;RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR;RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK;RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK;  and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have aglycine-cysteine (GC) attached to the C-terminus of the peptide, or aglycine-cysteine-glycine (GCG) attached to the N-terminus.

85. The method of any one of clauses 82 to 84 wherein the glycancomponent of the synthetic peptidoglycan is selected from the groupconsisting of dextran, chondroitin, chondroitin sulfate, dermatan,dermatan sulfate, heparan, heparin, keratin, keratan sulfate, andhyaluronic acid.

86. The method of any one of clauses 82 to 85 wherein the glycan isselected from the group consisting of chondroitin sulfate and keratansulfate.

87. The method of any one of clauses 82 to 86 wherein the syntheticpeptidoglycan is resistant to aggrecanase.

88. The method of any one of clauses 82 to 87 wherein the syntheticpeptidoglycan is administered to the patient by injection.

89. The method of clause 88 wherein the injection is subcutaneous.

90. The method of any one of clauses 82 to 89 wherein the defect is acosmetic defect.

91. A dermal filler comprising a hyaluronic acid-binding syntheticpeptidoglycan.

92. The dermal filler of clause 91 wherein the peptide component of thesynthetic peptidoglycan comprises an amino acid sequence of the formulaB1-X1-X2-X3 -X4-X5 -X6-X7-X8-B2,

wherein X8 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid, and

wherein X1-X8 are non-acidic amino acids.

93. The dermal filler of clause 91 or clause 92 wherein the peptidecomponent of the synthetic peptidoglycan comprises an amino acidsequence selected from the group consisting of:

GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK;SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP;SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR;RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR;RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK;RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK;  and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have aglycine-cysteine (GC) attached to the C-terminus of the peptide, or aglycine-cysteine-glycine (GCG) attached to the N-terminus.

94. The dermal filler of any one of clauses 91 to 93 further comprisinghyaluronic acid.

95. A method of reducing or preventing collagen degradation, said methodcomprising the steps of

contacting a hyaluronic acid-binding synthetic peptidoglycan withhyaluronic acid in the presence of collagen, and

reducing or preventing collagen degradation.

96. The method of clause 95 wherein the peptide component of thesynthetic peptidoglycan comprises an amino acid sequence of the formulaB1-X1-X2-X3-X4-X5-X6-X7-X8-B2,

wherein X8 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid, and

wherein X1-X8 are non-acidic amino acids.

97. The method of clause 95 or clause 96 wherein the peptide componentof the synthetic peptidoglycan comprises an amino acid sequence selectedfrom the group consisting of:

GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK;SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP;SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR;RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR;RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK;RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK;  and KRTMRPTRR.

98. The method of any one of clauses 95 to 97 wherein the glycancomponent of the synthetic peptidoglycan is selected from the groupconsisting of dextran, chondroitin, chondroitin sulfate, dermatan,dermatan sulfate, heparan, heparin, keratin, keratan sulfate, andhyaluronic acid.

99. The method of any one of clauses 95 to 98 wherein the glycan isselected from the group consisting of chondroitin sulfate and keratansulfate.

100. The method of any one of clauses 95 to 99 wherein the syntheticpeptidoglycan is resistant to aggrecanase.

101. The method of any one of clauses 95 to 100 wherein the rate ofhyaluronic acid degradation is reduced.

102. A method of increasing pore size in an engineered collagen matrix,said method comprising the steps of

combining collagen, hyaluronic acid, and a hyaluronic acid-bindingsynthetic peptidoglycan, and

increasing the pore size in the matrix.

103. The method of clause 102 wherein the peptide component of thesynthetic peptidoglycan comprises an amino acid sequence of the formulaB1-X1-X2-X3-X4-X5-X6-X7-X8-B2,

wherein X8 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid, and

wherein X1-X8 are non-acidic amino acids.

104. The method of clause 102 or clause 103 wherein the peptidecomponent of the synthetic peptidoglycan comprises an amino acidsequence selected from the group consisting of:

GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK;SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP;SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR;RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR;RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK;RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK;  and KRTMRPTRR.

105. The method of any one of clauses 102 to 104 wherein the glycancomponent of the synthetic peptidoglycan is selected from the groupconsisting of dextran, chondroitin, chondroitin sulfate, dermatan,dermatan sulfate, heparan, heparin, keratin, keratan sulfate, andhyaluronic acid.

106. The method of any one of clauses 102 to 105 wherein the glycan isselected from the group consisting of chondroitin sulfate and keratansulfate.

107. The method of any one of clauses 102 to 106 wherein the syntheticpeptidoglycan is resistant to aggrecanase.

108. The method of any one of clauses 102 to 107 wherein the matrix issterilized.

109. The method of any one of clauses 102 to 108 wherein the matrixfurther comprises chondrocytes or stem cells.

110. The method of clause 109 wherein the stem cells are selected fromthe group consisting of osteoblasts, osteogenic cells, and mesenchymalstem cells.

111. The method of any one of clauses 102 to 110 wherein the matrixfurther comprises one or more nutrients.

112. The method of any one of clauses 102 to 111 wherein the matrixfurther comprises one or more growth factors.

113. A method of reducing or preventing chondroitin sulfate degradation,said method comprising the steps of

contacting a hyaluronic acid-binding synthetic peptidoglycan withhyaluronic acid in the presence of collagen, and

reducing or preventing chondroitin sulfate degradation.

114. The method of clause 113 wherein the peptide component of thesynthetic peptidoglycan comprises an amino acid sequence of the formulaB1-X1-X2-X3-X4-X5-X6-X7-X8-B2,

wherein X8 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid, and

wherein X1-X8 are non-acidic amino acids.

115. The method of clause 113 or clause 114 wherein the peptidecomponent of the synthetic peptidoglycan comprises an amino acidsequence selected from the group consisting of:

GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK;SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP;SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR;RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR;RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK;RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK;  and KRTMRPTRR.

116. The method of any one of clauses 113 to 115 wherein the glycancomponent of the synthetic peptidoglycan is selected from the groupconsisting of dextran, chondroitin, chondroitin sulfate, dermatan,dermatan sulfate, heparan, heparin, keratin, keratan sulfate, andhyaluronic acid.

117. The method of any one of clauses 113 to 116 wherein the glycan isselected from the group consisting of chondroitin sulfate and keratansulfate.

118. The method of any one of clauses 113 to 117 wherein the syntheticpeptidoglycan is resistant to aggrecanase.

119. The method of any one of clauses 113 to 118 wherein the rate ofchondroitin sulfate degradation is reduced.

120. The synthetic peptidoglycan, compound, engineered collagen matrix,composition, additive, method, or dermal filler of any of the precedingclauses wherein the peptide component of the synthetic peptidoglycan hasa glycine-cysteine (GC) attached to the C-terminus of the peptide.

121. The synthetic peptidoglycan, compound, engineered collagen matrix,composition, additive, method, or dermal filler of any of the precedingclauses wherein the peptide component of the synthetic peptidoglycan hasa glycine-cysteine-glycine (GCG) attached to the N-terminus of thepeptide. p 122. The synthetic peptidoglycan, compound, engineeredcollagen matrix, composition, additive, method, or dermal filler of anyof the preceding clauses wherein the synthetic peptidoglycan isresistant to matrix metallo proteases.

123. The synthetic peptidoglycan, compound, engineered collagen matrix,composition, additive, method, or dermal filler of clause 122 whereinthe matrix metallo protease is aggrecanase.

124. The synthetic peptidoglycan, compound, engineered collagen matrix,composition, additive, method, or dermal filler of any of the precedingclauses wherein the dosage of the synthetic peptidoglycan is in aconcentration ranging from about 0.01 uM to about 100 uM.

125. The synthetic peptidoglycan, compound, engineered collagen matrix,composition, additive, method, or dermal filler of any of the precedingclauses wherein the dosage of the synthetic peptidoglycan is in aconcentration ranging from about 0.1 uM to about 10 uM.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a reaction schematic for the production of an embodiment ofthe hyaluronic acid-binding synthetic peptidoglycan. Reaction steps aredetailed in bold font.

FIG. 2 shows a standard curve of N-[β-Maleimidopropionic acid]hydrazide,trifluoroacetic acid salt (hereinafter “BMPH”) absorbance (215 nm) basedon amount (mg) of BMPH injected. The standard curve was used todetermine the amount of BMPH consumed during the coupling reaction.

FIG. 3 shows binding of the hyaluronic acid-binding syntheticpeptidoglycan to the immobilized hyaluronic acid (HA). Nine HA-bindingpeptides (e.g., GAHWQFNALTVRGGGC; hereinafter “GAH” or “mAGC”) wereattached to the functionalized glycosaminoglycan (e.g., chondroitinsulfate, hereinafter “CS”) backbone. Concentrations of syntheticpeptidoglycans were increased from 0.01 μM to 100 μM.

FIG. 4 shows HA binding of the synthetic peptidoglycan as determined byrheo logical frequency sweep (Panel A). The storage modulus of the HAmixtures was analyzed at an oscillatory frequency of 5.012 Hz. At thisfrequency, a noticeable load was provided while the integrity of the HAchains was maintained. Statistical analysis (α=0.05) showed that HA+CSand HA were significantly different (denoted *), and that HA+10.5 GAH-CSand HA+CS were significantly different (denoted **). Panel B is analternative representation of the same data shown in Panel A.

FIG. 5 shows quatification of the turbidity of the collagen type I plustreatment groups during collagen fibril formation. The absorbance at 313nm was measured every 3 minutes. After one hour (i.e., timepont number20), all treatment groups had completely formed networks. No significantdifferences (α=0.05) existed between treatment groups with respect tothe maximum absorbance or the time to half maximum absorbance.

FIG. 6 shows the compressive engineering stress withstood by thecollagen gels based on an applied engineering strain of 1% per second.Statistical analysis (α=0.05) demonstrated that the addition of 10.5GAH-CS resulted in a significant difference in peak engineering stress,in addition to the engineering stress analyzed at engineering strains of5%, 7.5%, and 10%.

FIG. 7 shows the storage modulus of collagen mixtures measured at anoscillatory frequency of 0.5012 Hz. Statistical analysis (α=0.05)demonstrated that the addition of 10.5 GAH-CS resulted in a significantincrease in the storage modulus of the collagen gel (denoted *).

FIG. 8 shows the percent degradation of HA mixtures due to the additionof hyaluronidase to the mixtures (Panel A). The percent degradation wasdetermined by the changes in the dynamic viscosity of the HA mixtures.Dynamic viscosity measurements were initially taken of the mixtures, andserved as the baseline from which the percent degradations werecalculated. The 0 hour timepoint was taken after the addition ofhyaluronidase, the sufficient mixing of the samples, and the pipettingonto the rheometer stage, and approximately 2 minutes passed between theaddition of the hyaluronidase and the measurement of the dynamicviscosity. Statistical analysis demonstrated significant differences(α=0.05) in the percent degradation for the 10.5 GAH-CS sample at boththe 0 hour and 2 hour timepoints. Panel B shows the same datarepresented as normalized dynamic viscosity (mean±SE, n=3) of HAmixtures due to the addition of hyaluronidase. Dynamic viscositymeasurements were initially taken of the mixtures before hyaluronidasewas added, and these values served as the baseline from which thenormalized dynamic viscosities were calculated. The normalized dynamicviscosities were determined by taking each measured dynamic viscosityafter the addition of hyaluronidase and dividing this value by theinitial dynamic viscosity of that sample. Statistical analysis (α=0.05)was conducted, and significant differences were seen in the normalizeddegradation for the 10.5 GAH-CS sample at both the 0 hr and 2 hrtimepoints.

FIG. 9 shows representative cryo-SEM images (10,000× magnification with5 μm scale bar) of the CI scaffold associated with each cartilage ECMreplicate. Panel A represents the CI control. Panel B representsCI+HA+CS. Panel C represents CI+HA+10.5 GAH-CS.

FIG. 10 shows the percent degradation (mean±SE, n=3) of CI in ECMreplicates exposed to MMP-I throughout a 50 hr duration. Statisticalanalysis (p<0.05) of the different treatments revealed that all threetreatments (CI control, CI+HA+CS, and CI+HA+10.5 GAH-CS) weresignificantly different from each other.

FIG. 11 shows the cumulative chondroitin sulfate (CS) loss over aneight-day culture period in media stimulated with and without IL-1β. CSloss was measured by a DMMB assay. The addition of mAGC had asignificant effect on loss of CS from the scaffolds (p<0.001). **denotes statistical significance between scaffold prepared withoutaggrecan mimic and those prepared with mAGC. + denotes statisticalsignificance between scaffold treated with and without IL-1β (p<0.05).Bars represent average ±SEM (n=3).

FIG. 12 shows the cumulative collagen breakdown over an eight-dayculture period in media stimulated with and without IL-1β. Collagenbreakdown was measured by a Sircol assay. The addition of aggrecan mimichad a significant effect on loss of collagen from the scaffolds(p<0.02). ** denotes statistical significance between scaffold preparedwithout aggrecan mimic and those prepared with mAGC. + denotesstatistical significance between scaffold treated with and without IL-1β(p<0.05). Bars represent average ±SEM (n=3).

FIG. 13 represents a platform to study the efficacy of the peptidoglycanex vivo. 0.5% trypsin was used to remove native aggrecan from bovinecartilage explants. Removal of aggrecan was confirmed by DMMB assay.Graphs represent the amount of aggrecan removed compared to positivecontrol.

FIG. 14 shows an assay to monitor peptidoglycan diffusion through thecartilage matrix. The Y-axis represents the difference in DMMB assayabsorbance values read from aggrecan-depleted cartilage plugs treatedwith/without peptidoglycan. The X-axis represents the distance from thearticular surface of cartilage to subchondral bone. Bars representaverage difference ±SEM (n=3).

FIG. 15 shows Safranin O and Avidin-Biotin stains of bovine cartilageexplants. A midsagittal cut was made through the matrix and probed forresidual aggrecan (top panel, dark staining) and biotin (bottom panel,dark staining) respectively. Collagen type II binding peptidoglycan[WYRGRLGC; “mAG(II)C”] was diffused through the explant. Highermagnification (20×m) of this tissue slice indicated that mAG(II)Cpenetrates approximately 200 um into tissue.

FIG. 16 shows Avidin-Biotin stains of cartilage explants. Peptidoglycans(mAG(II)C and mAGC) were diffused through the cartilage explant. Imagesindicate depth of penetration of each (dark staining).

FIG. 17 shows that the addition of peptidoglycans in aggrecan depleted(AD) explants increased compressive stiffness. Addition of the HAbinding peptidoglycan (mAGC) significantly restored stiffness ofcartilage explants to a higher extent as compared to the collagen typeTI binding peptidoglycan (mAG(TI)C). Significance, denoted as *,specified an increase in compressive stiffness between AD and AD+mACGaugmented explants (p<0.005). Data is presented as mean±SEM (n=5).

FIG. 18 (A) shows a schematic representation of probe bound to MMP-13.BHQ-3 black hole quencher 3 and CY5.5 absorbed and emitted at 695 nmrespectively. Arrow and italics indicate the cleavage site. (B) showsthe concentration profile of probe activity with and without MMP-13:Left, fluorescence imaging sections of 96-well microplate; Right,recovery of fluorescence emission intensity (695 nm).

FIG. 19 shows the extent of inflammation indicated by the MMP-13 probein Sprague-Dawley rats treated with and without peptidoglycan at four,six and eight weeks post surgery.

FIG. 20 shows a x-ray images of Sprague-Dawley rat knee joints showinginjured knee 6 weeks and 8 weeks following OA induction (Panels A and D,respectively), injured knee with peptidoglycan treatment (Panels B andE, respectively), and normal knee (Panel C) six weeks afterosteoarthritis induction surgery.

FIG. 21 shows microCT of Sprague-Dawley rats indicating re-growth of newcartilage six and eight weeks after OA induction surgery. Injured knees6 weeks and 8 weeks following OA induction are shown in Panels A and D,respectively. Injured knees following peptidoglycan treatment are shownin Panels B and E, respectively. Normal knee is shown in panel C.

FIG. 22 shows that the addition of mAGC to collagen scaffolds increasedthe storage modulus and compressive stiffness. Frequency sweeps (A) oncollagen scaffolds indicated an increase in storage modulus over a rangeof 0.1-2.0 Hz. Similarly, compressive stiffness (B) showed an increasein values when the scaffold was prepared with the addition of mAGC.Significance is denoted as * (p<0.0001). Data is presented as mean ±SEM(n=5).

FIG. 23 shows cumulative chondroitin sulfate (CS) loss over an eight-dayculture period in media stimulated with and without IL-1β. CS loss wasmeasured by a DMMB assay. Scaffold compositions (A-H) are described inTable 3. The addition of mAGC had a significant effect on loss of CSfrom the scaffolds (p<0.001). * denotes statistical significance betweenscaffold A and C, and scaffold E and G. (p<0.05). Bars represent average±SEM (n=3).

FIG. 24 shows cumulative collagen breakdown over an eight-day cultureperiod in media stimulated with and without 1L-1β. Collagen breakdownwas measured by a Sircol assay. Scaffold compositions (A-H) aredescribed in Table 3. The addition of aggrecan mimic had a significanteffect on loss of collagen from the scaffolds (p<0.02). * denotesstatistical significance between scaffold A and C, and scaffold E and G.(p<0.05). Bars represent average ±SEM (n=3).

FIG. 25 shows real-time PCR analysis for aggrecan and collagen type IIexpressed by bovine chondrocytes cultured in unaligned (A) and aligned(B) collagen scaffolds. Values were normalized to endogenous GAPDHexpression. Addition of mAGC statistically changed aggrecan and collagentype II expression (p_(aggrecan)<0.02 and p_(collagen)<0.001)respectively. There was also a statistical difference in aggrecan andcollagen type II expression between unaligned and aligned scaffolds(p<0.001). Similarly, the aggrecan and collagen type II expressiondiffered between scaffolds treated with and without IL-1β (p<0.01).Scaffold compositions (A-H) are described in Table 3. Bars representaverage ±SEM (n=4).

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

As used herein, a “hyaluronic acid-binding synthetic peptidoglycan”means a synthetic peptide conjugated to a glycan where the peptidecomprises a hyaluronic acid-binding sequence. Various embodiments of theinvention are described herein as follows. In one embodiment describedherein, a hyaluronic acid-binding synthetic peptidoglycan is provided.The hyaluronic acid-binding synthetic peptidoglycan comprises asynthetic peptide conjugated to a glycan wherein the synthetic peptidecomprises a hyaluronic acid-binding sequence.

In another embodiment, a compound of the formula P_(n)G_(x) is describedwherein n is 1 to 20; wherein x is 1 to 20; wherein P is a syntheticpeptide of about 5 to about 40 amino acids comprising a hyaluronic acidbinding sequence; and wherein G is a glycan.

In yet another embodiment, a compound of the formula (P_(n)L) _(x)G isdescribed

wherein n is 1 to 20;

wherein x is 1 to 20;

wherein P is a synthetic peptide of about 5 to about 40 amino acidscomprising a hyaluronic acid binding sequence;

wherein L is a linker; and

wherein G is a glycan.

In another embodiment, a compound of the formula P(LG_(n))_(x) isdescribed

wherein n is 1 to 20;

wherein x is 1 to 20;

wherein P is a synthetic peptide of about 5 to about 40 amino acidscomprising a hyaluronic acid binding sequence;

wherein L is a linker; and wherein G is a glycan.

In yet another embodiment, a compound of the formula P_(n)G_(x) isdescribed

wherein n is MWG/1000;

wherein MWG is the molecular weight of G rounded to the nearest 1 kDa;

wherein x is 1 to 20;

wherein P is a synthetic peptide of about 5 to about 40 amino acidscomprising a hyaluronic acid binding sequence; and

wherein G is a glycan.

In another embodiment, a compound of the formula (P_(n)L)_(x)G isdescribed

wherein n is MWG/1000;

wherein MWG is the molecular weight of G rounded to the nearest 1 kDa;

wherein x is 1 to 20;

wherein P is a synthetic peptide of about 5 to about 40 amino acidscomprising a hyaluronic acid binding sequence;

wherein L is a linker; and

wherein G is a glycan.

For purposes of this disclosure, the hyaluronic acid-binding syntheticpeptidoglycans and compounds described in the preceding paragraphs arecollectively referred to as “hyaluronic acid-binding syntheticpeptidoglycans” or “synthetic peptidoglycans.”

In each of the above peptide embodiments, the synthetic peptidoglycanmay comprise 5-15 peptide molecules (n is 5-15), 5-20 peptide molecules(n is 5-20), 1-20 peptide molecules (n is 1-20), or 1-25 peptidemolecules (n is 1-25). In one embodiment, n is selected from the groupconsisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, and 25 peptide molecules.

In another illustrative embodiment described herein, an engineeredcollagen matrix is provided. The matrix comprises polymerized collagen,hyaluronic acid, and a hyaluronic acid-binding synthetic peptidoglycan.In another embodiment, a composition for in vitro culture ofchondrocytes or stem cells is provided. The composition comprises any ofthe hyaluronic acid-binding synthetic peptidoglycans described in thisdisclosure.

In another embodiment described herein, a method of increasing pore sizein an engineered collagen matrix is provided. The method comprises thesteps of combining collagen, hyaluronic acid, and a hyaluronicacid-binding synthetic peptidoglycan and increasing the pore size in thematrix.

In yet another illustrative embodiment, a method of decreasing cartilagewear or erosion in a patient is provided. The method comprises the stepof administering to the patient a hyaluronic acid-binding syntheticpeptidoglycan, wherein the synthetic peptidoglycan decreases wear orerosion of the cartilage. In one embodiment, the cartilage erosion orwear may be caused by arthritis. In one embodiment, the cartilageerosion or wear may be caused by aging, obesity, trauma or injury, ananatomic abnormality, genetic diseases, metabolic imbalances,inflammation, or the like.

In yet another illustrative embodiment, a method of treatment forarthritis in a patient is provided. The method comprises the step ofadministering to the patient a hyaluronic acid-binding syntheticpeptidoglycan, wherein the synthetic peptidoglycan reduces a symptomassociated with the arthritis.

In another illustrative embodiment, a method of reducing or preventinghyaluronic acid degradation in a patient is provided. The methodcomprises administering to the patient a hyaluronic acid-bindingsynthetic peptidoglycan.

In another illustrative embodiment, a method of reducing or preventingcollagen degradation is provided. The method comprises the steps ofcontacting a hyaluronic acid-binding synthetic peptidoglycan withhyaluronic acid in the presence of collagen, and reducing or preventingcollagen degradation.

In yet another illustrative embodiment, a method for correcting ormodifying a tissue defect in a patient is provided. The method comprisesadministering into the tissue defect a hyaluronic acid-binding syntheticpeptidoglycan wherein the defect is corrected or modified.

In another illustrative embodiment described herein, a dermal filler isprovided. The filler comprises a hyaluronic acid-binding syntheticpeptidoglycan. In one embodiment, the filler further compriseshyaluronic acid.

In yet another embodiment, an additive for a biomaterial cartilage orbone replacement composition is provided. The additive comprises ahyaluronic acid-binding synthetic peptidoglycan for addition to anexisting biomaterial cartilage or bone replacement material. In anotherembodiment described herein, a method of preparing a biomaterial or bonecartilage replacement is provided. The method comprises the step ofcombining the synthetic peptidoglycan and an existing biomaterial orbone cartilage replacement material.

In the various embodiments, the peptide component of the syntheticpeptidoglycan comprises an amino acid sequence of the formulaB1-X1-X2-X3-X4-X5-X6-X7-X8-B2,

wherein X8 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid, and

wherein X1-X8 are non-acidic amino acids.

In another embodiment, the peptide component of the syntheticpeptidoglycan can comprise or can be an amino acid sequence of theformula B1-X1-B2-X2-X3-X4-X5-X6-X7-X8-X9-B3,

wherein X9 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid,

wherein B3 is a basic amino acid, and

wherein X1-X9 are non-acidic amino acids.

In another embodiment, the synthetic peptide can comprise or can be anamino acid sequence of the formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2-X9-B3,

wherein X8 is present or is not present,

wherein B1 is a basic amino acid,

wherein B2 is a basic amino acid,

wherein B3 is a basic amino acid, and

wherein X1-X9 are non-acidic amino acids.

As used herein, a “basic amino acid” is selected from the groupconsisting of lysine, arginine, or histidine. As used herein, a“non-acidic amino acid” is selected from the group consisting ofalanine, arginine, asparagine, cysteine, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine.

In the various illustrative embodiments described herein, the peptidecomponent of the synthetic peptidoglycan can comprise an amino acidsequence selected from the group consisting of:

GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK;SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP;SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR;RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR;RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK;RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK;  and KRTMRPTRR.In each of the above peptide embodiments, the peptide may have aglycine-cysteine attached to the C-terminus of the peptide, and/or aglycine-cysteine-glycine (GCG) attached to the N-terminus of thepeptide. In various embodiments described herein, the peptide componentof the synthetic peptidoglycan comprises any amino acid sequencedescribed in the preceding paragraph or an amino acid sequence with 80%,85%, 90%, 95%, 98%, or 100% homology to any of these amino acidsequences.

Additional peptides that can be included as the peptide component of thehyaluronic acid-binding synthetic peptidoglycans include peptidesdescribed in Amemiya et al., Biochem. Biophys. Acta, vol. 1724, pp.94-99 (2005), incorporated herein by reference. These peptides have anArg-Arg motif and include peptides selected from the group consistingof:

RRASRSRGQVGL; GRGTHHAQKRRS; QPVRRLGTPVVG; ARRAEGKTRMLQ; PKVRGRRHQASG;SDRHRRRREADG; NQRVRRVKHPPG; RERRERHAVARHGPGLERDARNLARR;TVRPGGKRGGQVGPPAGVLHGRRARS; NVRSRRGHRMNS; DRRRGRTRNIGN; KTAGHGRRWSRN;AKRGEGRREWPR; GGDRRKAHKLQA; RRGGRKWGSFEG;  and RQRRRDLTRVEG.In each of the above peptide embodiments, the peptide may have aglycine-cysteine attached to the C-terminus of the peptide. In each ofthe above peptide embodiments, the peptide may have aglycine-cysteine-glycine (GCG) attached to the N-terminus of thepeptide. In various embodiments described herein, the peptide componentof the synthetic peptidoglycan comprises any amino acid sequencedescribed in the preceding paragraph or an amino acid sequence with 80%,85%, 90%, 95%, 98%, or 100% homology to any of these amino acidsequences.

In other embodiments, peptides described in Yang et al., EMBO Journal,vol. 13, pp. 286-296 (1994), incorporated herein by reference, andGoetinck et al., J. Cell. Biol., vol. 105, pp. 2403-2408 (1987),incorporated herein by reference, can be used in the hyaluronicacid-binding synthetic peptidoglycans described herein includingpeptides selected from the group consisting of RDGTRYVQKGEYR,HREARSGKYK, PDKKHKLYGV, and WDKERSRYDV. In each of these embodiments,the peptide may have a glycine-cysteine attached to the C-terminus ofthe peptide. In each of these embodiments, the peptide may have aglycine-cysteine-glycine (GCG) attached to the N-terminus of thepeptide. In other embodiments, the peptide component of the syntheticpeptidoglycan comprises an amino acid sequence with 80%, 85%, 90%, 95%,98%, or 100% homology to any of these amino acid sequences.

In various embodiments, the peptide component of the syntheticpeptidoglycan described herein can be modified by the inclusion of oneor more conservative amino acid substitutions. As is well-known to thoseskilled in the art, altering any non-critical amino acid of a peptide byconservative substitution should not significantly alter the activity ofthat peptide because the side-chain of the replacement amino acid shouldbe able to form similar bonds and contacts to the side chain of theamino acid which has been replaced. Non-conservative substitutions arepossible provided that these do not excessively affect the hyaluronicacid binding activity of the peptide.

As is well-known in the art, a “conservative substitution” of an aminoacid or a “conservative substitution variant” of a peptide refers to anamino acid substitution which maintains: 1) the secondary structure ofthe peptide; 2) the charge or hydrophobicity of the amino acid; and 3)the bulkiness of the side chain or any one or more of thesecharacteristics.

Illustratively, the well-known terminologies “hydrophilic residues”relate to serine or threonine. “Hydrophobic residues” refer to leucine,isoleucine, phenylalanine, valine or alanine, or the like. “Positivelycharged residues” relate to lysine, arginine, ornithine, or histidine.“Negatively charged residues” refer to aspartic acid or glutamic acid.Residues having “bulky side chains” refer to phenylalanine, tryptophanor tyrosine, or the like. A list of illustrative conservative amino acidsubstitutions is given in TABLE 1.

TABLE 1 For Amino Acid Replace With Alanine D-Ala, Gly, Aib, β-Ala,L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Orn D-Orn Asparagine D-Asn,Asp, D-Asp, Glu, D-Glu Gln, D- Gln Aspartic Acid D-Asp, D-Asn, Asn, Glu,D-Glu, Gln, D- Gln Cysteine D-Cys, S—Me-Cys, Met, D-Met, Thr, D- ThrGlutamine D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D- Asp Glutamic AcidD-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D- Gln Glycine Ala, D-Ala, Pro,D-Pro, Aib, β-Ala Isoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D- MetLeucine Val, D-Val, Met, D-Met, D-Ile, D-Leu, Ile Lysine D-Lys, Arg,D-Arg, Orn, D-Orn Methionine D-Met, S—Me-Cys, Ile, D-Ile, Leu, D-Leu,Val, D-Val Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D- TrpProline D-Pro Serine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys ThreonineD-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-Val Tyrosine D-Tyr, Phe,D-Phe, His, D-His, Trp, D- Trp Valine D-Val, Leu, D-Leu, Ile, D-Ile,Met, D- MetIn one embodiment, the conservative amino acid substitutions appicableto the molecules described herein do not alter the motifs that consistof the B1-X1-X2-X3-X4-X5-X6-X7-X8-B2 formula, theB1-X1-B2-X2-X3--X4-X5-X6-X7-X8-X9-B3 formula, theB1-X1-X2-X3-X4-X5-X6-X7-X8-B2-X9-B3 formula, or the Arg-Arg motif.

In various embodiments described herein, the glycan (e.g.glycosaminoglycan, abbreviated GAG, or polysaccharide) component of thesynthetic peptidoglycan described herein can be selected from the groupconsisting of dextran, chondroitin, chondroitin sulfate, dermatan,dermatan sulfate, heparan, heparin, keratin, keratan sulfate, andhyaluronic acid. In one embodiment, the glycan is selected from thegroup consisting of chondroitin sulfate and keratan sulfate. In anotherillustrative embodiment, the glycan is chondroitin sulfate.

In one embodiment described herein, the hyaluronic acid-bindingsynthetic peptidoglycan comprises (GAHWQFNALTVRGG)₁₀ conjugated tochondroitin sulfate wherein each peptide in the peptidoglycan moleculeis linked separately to chondroitin sulfate. In another embodimentdescribed herein, the hyaluronic acid-binding synthetic peptidoglycancomprises (GAHWQFNALTVRGGGC)₁₁ conjugated to chondroitin sulfate whereineach peptide in the peptidoglycan molecule is linked separately tochondroitin sulfate. In each of the above peptide embodiments, thepeptide number may be selected from the group consisting of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, and 25 peptide molecules.

In various embodiments described herein, the synthetic peptidoglycan isresistant to aggrecanase. An aggrecanase is characterized in the art asany enzyme known to cleave aggrecan.

In one illustrative aspect, the hyaluronic acid-binding syntheticpeptidoglycan may be sterilized. As used herein “sterilization” or“sterilize” or “sterilized” means disinfecting the hyaluronicacid-binding synthetic peptidoglycans by removing unwanted contaminantsincluding, but not limited to, endotoxins and infectious agents.

In various illustrative embodiments, the hyaluronic acid-bindingsynthetic peptidoglycan can be disinfected and/or sterilized usingconventional sterilization techniques including propylene oxide orethylene oxide treatment, gas plasma sterilization, gamma radiation(e.g., 1-4 Mrads gamma irradiation or 1-2.5 Mrads of gamma irradiation),electron beam, and/or sterilization with a peracid, such as peraceticacid. Sterilization techniques which do not adversely affect thestructure and biotropic properties of the hyaluronic acid-bindingsynthetic peptidoglycan can be used. In one embodiment, the hyaluronicacid-binding synthetic peptidoglycan can be subjected to one or moresterilization processes. In another illustrative embodiment, thehyaluronic acid-binding synthetic peptidoglycan is subjected to sterilefiltration. The hyaluronic acid-binding synthetic peptidoglycan may bewrapped in any type of container including a plastic wrap or a foilwrap, and may be further sterilized. The hyaluronic acid-bindingsynthetic peptidoglycan may be prepared under sterile conditions, forexample, by lyophilisation, which may readily be accomplished usingstandard techniques well-known to those skilled in the art.

In various embodiments described herein, the hyaluronic acid-bindingsynthetic peptidoglycans can be combined with minerals, amino acids,sugars, peptides, proteins, vitamins (such as ascorbic acid), orlaminin, collagen, fibronectin, hyaluronic acid, fibrin, elastin, oraggrecan, or growth factors such as epidermal growth factor,platelet-derived growth factor, transforming growth factor beta, orfibroblast growth factor, and glucocorticoids such as dexamethasone orviscoelastic altering agents, such as ionic and non-ionic water solublepolymers; acrylic acid polymers; hydrophilic polymers such aspolyethylene oxides, polyoxyethylene-polyoxypropylene copolymers, andpolyvinylalcohol; cellulosic polymers and cellulosic polymer derivativessuch as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethylcellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethyl cellulose, and etherified cellulose;poly(lactic acid), poly(glycolic acid), copolymers of lactic andglycolic acids, or other polymeric agents both natural and synthetic.

In various embodiments described herein, the peptide component of thesynthetic peptidoglycan is synthesized according to solid phase peptidesynthesis protocols that are well-known by persons of skill in the art.In one embodiment a peptide precursor is synthesized on a solid supportaccording to the well-known Fmoc protocol, cleaved from the support withtrifluoroacetic acid and purified by chromatography according to methodsknown to persons skilled in the art.

In various embodiments described herein, the peptide component of thesynthetic peptidoglycan is synthesized utilizing the methods ofbiotechnology that are well-known to persons skilled in the art. In oneembodiment a DNA sequence that encodes the amino acid sequenceinformation for the desired peptide is ligated by recombinant DNAtechniques known to persons skilled in the art into an expressionplasmid (for example, a plasmid that incorporates an affinity tag foraffinity purification of the peptide), the plasmid is transfected into ahost organism for expression, and the peptide is then isolated from thehost organism or the growth medium according to methods known by personsskilled in the art (e.g., by affinity purification). Recombinant DNAtechnology methods are described in Sambrook et al., “Molecular Cloning:A Laboratory Manual”, 3rd Edition, Cold Spring Harbor Laboratory Press,(2001), incorporated herein by reference, and are well-known to theskilled artisan.

In various embodiments described herein, the peptide component of thehyaluronic acid-binding synthetic peptidoglycan is conjugated to aglycan by reacting a free amino group of the peptide with an aldehydefunction of the glycan in the presence of a reducing agent, utilizingmethods known to persons skilled in the art, to yield the peptide glycanconjugate. In one embodiment an aldehyde function of the glycan (e.g.polysaccharide or glycosaminoglycan) is formed by reacting the glycanwith sodium metaperiodate according to methods known to persons skilledin the art.

In one embodiment, the peptide component of the synthetic peptidoglycanis conjugated to a glycan by reacting an aldehyde function of the glycanwith 3-(2-pyridyldithio)propionyl hydrazide (PDPH) to form anintermediate glycan and further reacting the intermediate glycan with apeptide containing a free thiol group to yield the peptide glycanconjugate. In yet another embodiment, the sequence of the peptidecomponent of the synthetic peptidoglycan may be modified to include aglycine-cysteine segment to provide an attachment point for a glycan ora glycan-linker conjugate. In any of the embodiments described herein,the crosslinker can be N[β-Maleimidopropionic acid]hydrazide (BMPH).

Although specific embodiments have been described in the precedingparagraphs, the hyaluronic acid-binding synthetic peptidoglycansdescribed herein can be made by using any art-recognized method forconjugation of the peptide to the glycan (e.g. polysaccharide orglycosaminoglycan). This can include covalent, ionic, or hydrogenbonding, either directly or indirectly via a linking group such as adivalent linker. The conjugate is typically formed by covalent bondingof the peptide to the glycan through the formation of amide, ester orimino bonds between acid, aldehyde, hydroxy, amino, or hydrazo groups onthe respective components of the conjugate. All of these methods areknown in the art or are further described in the Examples section ofthis application or in Hermanson G. T., Bioconjugate Techniques,Academic Press, pp. 169-186 (1996), incorporated herein by reference.The linker typically comprises about 1 to about 30 carbon atoms, moretypically about 2 to about 20 carbon atoms. Lower molecular weightlinkers (i.e., those having an approximate molecular weight of about 20to about 500) are typically employed.

In addition, structural modifications of the linker portion of theconjugates are contemplated herein. For example, amino acids may beincluded in the linker and a number of amino acid substitutions may bemade to the linker portion of the conjugate, including but not limitedto naturally occurring amino acids, as well as those available fromconventional synthetic methods. In another aspect, beta, gamma, andlonger chain amino acids may be used in place of one or more alpha aminoacids. In another aspect, the linker may be shortened or lengthened,either by changing the number of amino acids included therein, or byincluding more or fewer beta, gamma, or longer chain amino acids.Similarly, the length and shape of other chemical fragments of thelinkers described herein may be modified.

In various embodiments described herein, the linker may include one ormore bivalent fragments selected independently in each instance from thegroup consisting of alkylene, heteroalkylenc, cycloalkylene,cycloheteroalkylene, arylenc, and heteroarylenc each of which isoptionally substituted. As used herein heteroalkylene represents a groupresulting from the replacement of one or more carbon atoms in a linearor branched alkylene group with an atom independently selected in eachinstance from the group consisting of oxygen, nitrogen, phosphorus andsulfur. In an alternative embodiment, a linker is not present.

In one embodiment described herein, an engineered collagen matrix isprovided. The previously described embodiments of the hyaluronicacid-binding synthetic peptidoglycan are applicable to the engineeredcollagen matrix described herein. In one embodiment, the engineeredcollagen matrix comprises polymerized collagen, hyaluronic acid, and ahyaluronic acid-binding synthetic peptidoglycan. In one embodiment, theengineered collagen matrix comprises polymerized collagen and ahyaluronic-binding synthetic peptidoglycan. In various illustrativeembodiments, crosslinking agents, such as carbodiimides, aldehydes,lysl-oxidase, N-hydroxysuccinimide esters, imidoesters, hydrazides, andmaleimides, as well as various natural crosslinking agents, includinggenipin, and the like, can be added before, during, or afterpolymerization of the collagen in solution.

In various illustrative embodiments, the collagen used herein to preparean engineered collagen matrix may be any type of collagen, includingcollagen types I to XXVIII, alone or in any combination, for example,collagen types I, II, III, and/or IV may be used. In some embodiments,the collagen used to prepare an engineered collagen matrix is selectedfrom the group consisting of type I collagen, type II collagen, type IIIcollagen, type IV collagen, type IX collagen, type XI collagen, andcombinations thereof. In one embodiment, the engineered collagen matrixis formed using commercially available collagen (e.g., Sigma, St. Louis,Mo.). In an alternative embodiment, the collagen can be purified fromsubmucosa-containing tissue material such as intestinal, urinarybladder, or stomach tissue. In a further embodiment, the collagen can bepurified from tail tendon. In an additional embodiment, the collagen canbe purified from skin. In various aspects, the collagen can also containendogenous or exogenously added non-collagenous proteins in addition tothe collagen-binding synthetic peptidoglycans, such as fibronectin orsilk proteins, glycoproteins, and polysaccharides, or the like. Theengineered collagen matrices prepared by the methods described hereincan be in the form of a tissue graft (e.g., in the form of a gel) whichcan assume the characterizing features of the tissue(s) with which theyare associated at the site of implantation or injection. In oneembodiment, the engineered collagen matrix is a tissue graft that can beimplanted into a patient. In another embodiment, the engineered collagenmatrix can be administered to a patient by injection. In eitherembodiment, the matrix can be in the form of a gel or a powder, forexample.

In one embodiment, the collagen in the engineered collagen matrixcomprises about 40 to about 90 dry weight (wt) % of the matrix, about 40to about 80 dry wt % of the matrix, about 40 to about 70 dry wt % of thematrix, about 40 to about 60 dry wt % of the matrix, about 50 to about90 dry wt % of the matrix, about 50 to about 80 dry wt % of the matrix,about 50 to about 75 dry wt % of the matrix, about 50 to about 70 dry wt% of the matrix, or about 60 to about 75 dry wt % of the matrix. Inanother embodiment, the collagen in the engineered collagen matrixcomprises about 90 dry wt %, about 85 dry wt %, about 80 dry wt %, about75 dry wt %, about 70 dry wt %, about 65 dry wt %, about 60 dry wt %,about 50 dry wt %, about 45 dry wt %, about 40 dry wt %, or about 30 drywt % of the matrix.

In one embodiment, the final collagen concentration of the matrix in gelform is about 0.5 to about 6 mg per mL, about 0.5 to about 5 mg per mL,about 0.5 to about 4 mg per mL, about 1 to about 6 mg per mL, about 1 toabout 5 mg per mL, or about 1 to about 4 mg per mL. In one embodiment,the final collagen concentration of the matrix is about 0.5 mg per mL,about 1 mg per mL, about 2 mg per mL, about 3 mg per mL, about 4 mg permL, or about 5 mg per mL.

In one embodiment, the hyaluronic acid-binding synthetic peptidoglycanin the engineered collagen matrix comprises about 2 to about 60 dryweight (wt) % of the matrix, about 2 to about 50 dry wt % of the matrix,about 5 to about 50 dry wt % of the matrix, about 10 to about 50 dry wt% of the matrix, about 10 to about 20 dry wt % of the matrix, about 10to about 30 dry wt % of the matrix, about 10 to about 25 dry wt % of thematrix, about 15 to about 30 dry wt % of the matrix, or about 15 toabout 45 dry wt % of the matrix. In another embodiment, the hyaluronicacid-binding synthetic peptidoglycan in the engineered collagen matrixcomprises about 2 dry wt %, about 5 dry wt %, about 10 dry wt %, about15 dry wt %, about 20 dry wt %, about 25 dry wt %, about 30 dry wt %,about 35 dry wt %, about 40 dry wt %, about 45 dry wt %, or about 50 drywt % of the matrix

In another embodiment, the engineered collagen matrix compriseshyaluronic acid and the hyaluronic acid in the engineered collagenmatrix comprises about 2 to about 60 dry weight (wt) % of the matrix,about 2 to about 50 dry wt % of the matrix, about 5 to about 50 dry wt %of the matrix, about 10 to about 50 dry wt % of the matrix, about 10 toabout 20 dry wt % of the matrix, about 10 to about 30 dry wt % of thematrix, about 10 to about 25 dry wt % of the matrix, about 15 to about30 dry wt % of the matrix, or about 15 to about 45 dry wt % of thematrix. In another embodiment, the hyaluronic acid in the engineeredcollagen matrix comprises about 2 dry wt %, about 5 dry wt %, about 10dry wt %, about 15 dry wt %, about 20 dry wt %, about 25 dry wt %, about30 dry wt %, about 35 dry wt %, about 40 dry wt %, about 45 dry wt %, orabout 50 dry wt % of the matrix.

In one embodiment, the engineered collagen matrix comprises hyaluronicacid and a hyaluronic acid-binding synthetic peptidoglycan. Thehyaluronic acid and hyaluronic acid-binding synthetic peptidoglycan inthe engineered collagen matrix comprise about 10 to about 60 dry weight(wt) % of the matrix, about 20 to about 60 dry wt % of the matrix, about30 to about 60 dry wt % of the matrix, about 40 to about 60 dry wt % ofthe matrix, about 10 to about 50 dry wt % of the matrix, about 20 toabout 50 dry wt % of the matrix, about 25 to about 50 dry wt % of thematrix, about 30 to about 50 dry wt % of the matrix, or about 25 toabout 40 dry wt % of the matrix. In another embodiment, the hyaluronicacid and hyaluronic acid-binding synthetic peptidoglycan in theengineered collagen matrix comprises about 10 dry wt %, about 15 dry wt%, about 20 dry wt %, about 25 dry wt %, about 30 dry wt %, about 35 drywt %, about 40 dry wt %, about 50 dry wt %, about 55 dry wt %, about 60dry wt %, or about 70 dry wt % of the matrix.

In one illustrative aspect, the engineered collagen matrix may besterilized. As used herein “sterilization” or “sterilize” or“sterilized” means disinfecting the matrix by removing unwantedcontaminants including, but not limited to, endotoxins, nucleic acidcontaminants, and infectious agents.

In various illustrative embodiments, the engineered collagen matrix canbe disinfected and/or sterilized using conventional sterilizationtechniques including glutaraldehyde tanning, formaldehyde tanning atacidic pH, propylene oxide or ethylene oxide treatment, gas plasmasterilization, gamma radiation (e.g., 1-4 Mrads gamma irradiation or1-2.5 Mrads of gamma irradiation), electron beam, and/or sterilizationwith a peracid, such as peracetic acid. Sterilization techniques whichdo not adversely affect the structure and biotropic properties of thematrix can be used. In one embodiment, the engineered collagen matrixcan be subjected to one or more sterilization processes. In illustrativeembodiments, the collagen in solution, prior to polymerization, can alsobe sterilized or disinfected. The engineered collagen matrix may bewrapped in any type of container including a plastic wrap or a foilwrap, and may be further sterilized.

In any of these embodiments the engineered collagen matrix may furthercomprise an exogenous population of cells. The added population of cellsmay comprise one or more cell populations. In various embodiments, thecell populations comprise a population of non-keratinized or keratinizedepithelial cells or a population of cells selected from the groupconsisting of endothelial cells, mesodermally derived cells, mesothelialcells, synoviocytes, neural cells, glial cells, osteoblasts,fibroblasts, chondrocytes, tenocytes, smooth muscle cells, skeletalmuscle cells, cardiac muscle cells, multi-potential progenitor cells(e.g., stem cells, including bone marrow progenitor cells), andosteogenic cells. In some embodiments, the population of cells isselected from the group consisting of chondrocytes and stem cells. Insome embodiments, the stem cells are selected from the group consistingof osteoblasts, osteogenic cells, and mesenchymal stem cells. In variousembodiments, the engineered collagen matrix can be seeded with one ormore cell types in combination.

In various aspects, the engineered collagen matrices or engineered graftconstructs of the present invention can be combined with nutrients,including minerals, amino acids, sugars, peptides, proteins, vitamins(such as ascorbic acid), or laminin, fibronectin, hyaluronic acid,fibrin, elastin, or aggrecan, or growth factors such as epidermal growthfactor, platelet-derived growth factor, transforming growth factor beta,or fibroblast growth factor, and glucocorticoids such as dexamethasoneor viscoelastic altering agents, such as ionic and non-ionic watersoluble polymers; acrylic acid polymers; hydrophilic polymers such aspolyethylene oxides, polyoxyethylene-polyoxypropylene copolymers, andpolyvinylalcohol; cellulosic polymers and cellulosic polymer derivativessuch as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethylcellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethyl cellulose, and etherified cellulose;poly(lactic acid), poly(glycolic acid), copolymers of lactic andglycolic acids, or other polymeric agents both natural and synthetic. Inother illustrative embodiments, cross-linking agents, such ascarbodiimides, aldehydes, lysl-oxidase, N-hydroxysuccinimide esters,imidoesters, hydrazides, and maleimides, as well as natural crosslinkingagents, including genipin, and the like can be added before, concurrentwith, or after the addition of cells.

As discussed above, in accordance with one embodiment, cells can beadded to the engineered collagen matrices or the engineered graftconstructs after polymerization of the collagen or during collagenpolymerization. The engineered collagen matrices comprising the cellscan be subsequently injected or implanted in a host for use asengineered graft constructs. In another embodiment, the cells on orwithin the engineered collagen matrices can be cultured in vitro, for apredetermined length of time, to increase the cell number or to inducedesired remodeling prior to implantation or injection into a patient.

In one embodiment described herein, a composition for in vitro cultureof chondrocytes or stem cells is provided (i.e., for in vitro culture ofcells without subsequent implantation or injection into a patient). Thecomposition for in vitro culture comprises a hyaluronic acid-bindingsynthetic peptidoglycan. The previously described embodiments of thehyaluronic acid-binding synthetic peptidoglycan are applicable to thecomposition for in vitro culture described herein.

In various aspects, the composition for in vitro culture of the presentinvention can be combined with nutrients, including minerals, aminoacids, sugars, peptides, proteins, vitamins (such as ascorbic acid), orlaminin, fibronectin, hyaluronic acid, fibrin, elastin, or aggrecan, orgrowth factors such as epidermal growth factor, platelet-derived growthfactor, transforming growth factor beta, or fibroblast growth factor,and glucocorticoids such as dexamethasone.

In some embodiments, the composition for in vitro culture includes stemcells selected from the group consisting of osteoblasts, osteogeniccells, and mesenchymal stem cells. In various embodiments, thecomposition for in vitro culture can be seeded with one or more celltypes in combination.

In one illustrative aspect, the composition for in vitro culture may besterilized. As used herein “sterilization” or “sterilize” or“sterilized” means disinfecting the composition by removing unwantedcontaminants including, but not limited to, endotoxins, nucleic acidcontaminants, and infectious agents. The sterilization procedures,methods and embodiments provided in the preceding paragraphs are alsoapplicable to the composition for in vitro culture described herein. Thein vitro culture composition may be used to expand populations of cellsfor implantation or injection into a patient.

In one embodiment described herein, an additive for a biomaterialcartilage replacement composition is provided. The additive comprises ahyaluronic acid-binding synthetic peptidoglycan for addition to anexisting biomaterial cartilage replacement material. The previouslydescribed embodiments of the hyaluronic acid-binding syntheticpeptidoglycan are applicable to the additive described herein.

As used herein, the phrase “existing biomaterial cartilage replacementmaterial” means a biologically compatible composition that can beutilized for replacement of damaged, defective, or missing cartilage inthe body. Various types of existing biomaterial cartilage replacementcompositions are well-known in the art and are contemplated. Forexample, existing biomaterial cartilage or bone replacement compositionsinclude the DeNovo® NT Natural Tissue Graft (Zimmer), MaioRegen™ (JRILimited), or the collection of cryopreserved osteoarticular tissuesproduced by Biomet.

In one embodiment, a method of preparing a biomaterial or bone cartilagereplacement is provided. The method comprises the step of combining thesynthetic peptidoglycan and an existing biomaterial or bone cartilagereplacement material. The previously described embodiments of thehyaluronic acid-binding synthetic peptidoglycan are applicable to themethod described herein.

In one embodiment, a method of treatment for arthritis in a patient isprovided. The method comprises the step of administering to the patienta hyaluronic acid-binding synthetic peptidoglycan, wherein the syntheticpeptidoglycan reduces one or more symptoms associated with arthritis.The previously described embodiments of the hyaluronic acid-bindingsynthetic peptidoglycan are applicable to the method described herein.

In various embodiments, the synthetic peptidoglycan used in the methodof treatment for arthritis reduces one or more symptoms associated witharthritis. Various symptoms are known in the art to be associated witharthritis, including but not limited to pain, stiffness, tenderness,inflammation, swelling, redness, warmth, and decreased mobility. Thesymptoms of arthritis may be present in a joint, a tendon, or otherparts of the body. As used herein, “reducing” means preventing orcompletely or partially alleviating a symptom of arthritis.

In various embodiments, the arthritis is osteoarthritis or rheumatoidarthritis. The pathogenesis and clinical symptoms of osteoarthritis andrheumatoid arthritis are well-known in the art. In one embodiment ofthis method, the synthetic peptidoglycan acts as a lubricant followingadministration or prevents loss of cartilage. In another embodiment, thesynthetic peptidoglycan prevents articulation of bones in the patient.For example, the synthetic peptidoglycan inhibits bone on bonearticulation in a patient with reduced or damaged cartilage.

In one embodiment, a method of reducing or preventing degradation of ECMcomponents in a patient is provided. For example, a method of reducingor preventing degradation of ECM components in the cartilage of apatient is provided. The method comprises administering to the patient ahyaluronic acid-binding synthetic peptidoglycan. The previouslydescribed embodiments of the hyaluronic acid-binding syntheticpeptidoglycan are applicable to the method described herein. In oneembodiment, the synthetic peptidoglycan is resistant to matrix metalloproteases, e.g., an aggrecanase.

In another embodiment, a method of reducing or preventing hyaluronicacid degradation in a patient is provided. The method comprisesadministering to the patient a hyaluronic acid-binding syntheticpeptidoglycan. The previously described embodiments of the hyaluronicacid-binding synthetic peptidoglycan are applicable to the methoddescribed herein.

In another embodiment, a method of reducing or preventing collagendegradation is provided. The method comprises the steps of contacting ahyaluronic acid-binding synthetic peptidoglycan with hyaluronic acid inthe presence of collagen, and reducing or preventing collagendegradation. The previously described embodiments of the hyaluronicacid-binding synthetic peptidoglycan arc applicable to the methoddescribed herein.

In another embodiment, a method of reducing or preventing chondroitinsulfate degradation is provided. The method comprises the steps ofcontacting a hyaluronic acid-binding synthetic peptidoglycan withhyaluronic acid in the presence of collagen, and reducing or preventingchondroitin sulfate degradation. The previously described embodiments ofthe hyaluronic acid-binding synthetic peptidoglycan are applicable tothe method described herein.

“Reducing” ECM component degradation, e.g., hyaluronic acid, collagen,or chondroitin sulfate degradation, means completely or partiallyreducing degradation of hyaluronic acid, collagen, or chondroitinsulfate, respectively.

In one embodiment, reducing hyaluronic acid degradation in a patientmeans reducing the rate of hyaluronic acid degradation. For example,FIG. 8 described in the Examples section of the application shows thatthe rate of hyaluronic acid degradation in a mixture of hyaluronic acidand a hyaluronic acid-binding synthetic peptidoglycan is significantlyreduced upon addition of the synthetic peptidoglycan.

In one embodiment, reducing collagen degradation means reducing the rateof collagen degradation. For example, FIG. 10 described in the Examplessection of the application shows that the rate of collagen degradationin the presence of hyaluronic acid and a hyaluronic acid-bindingsynthetic peptidoglycan is significantly reduced upon addition of thesynthetic peptidoglycan.

In one embodiment, reducing chondroitin sulfate degradation meansreducing the rate of chondroitin sulfate degradation. For example, FIG.11 described in the Examples section of the application shows that therate of chondroitin sulfate degradation in the presence of a hyaluronicacid-binding synthetic peptidoglycan is significantly reduced uponaddition of the synthetic peptidoglycan.

In one embodiment described herein, a method for correcting or modifyinga tissue defect in a patient is provided. The method comprisesadministering into the tissue defect hyaluronic acid and a hyaluronicacid-binding synthetic peptidoglycan wherein the defect is corrected ormodified. The previously described embodiments of the hyaluronicacid-binding synthetic peptidoglycan are applicable to the methoddescribed herein. In one embodiment, the tissue defect is a cosmeticdefect.

The following embodiments are applicable to methods described hereinwhere the hyaluronic acid-binding synthetic peptidoglycan isadministered to a patient. In various embodiments, the hyaluronicacid-binding synthetic peptidoglycan can be injected or implanted (e.g.,incorporated in a cartilage repair composition or device). In someembodiments described herein, the injection is an intraarticularinjection. In another embodiment described herein, the injection is intoa joint capsule of the patient. In other embodiments, the injection is asubcutaneous injection, as in the case of dermal fillers. Suitable meansfor injection include a needle (including microneedle) injector or adevice for infusion.

In an illustrative embodiment, pharmaceutical formulations for use withhyaluronic acid-binding synthetic peptidoglycans for administration to apatient comprise: a) a pharmaceutically active amount of the hyaluronicacid-binding synthetic peptidoglycan; b) a pharmaceutically acceptablepH buffering agent to provide a pH in the range of about pH 4.5 to aboutpH 9; c) an ionic strength modifying agent in the concentration range ofabout 0 to about 300 millimolar; and d) water soluble viscositymodifying agent in the concentration range of about 0.25% to about 10%total formula weight or any individual component a), b), c), or d) orany combinations of a), b), c) and d).

In various embodiments described herein, the pH buffering agents arethose agents known to the skilled artisan and include, for example,acetate, borate, carbonate, citrate, and phosphate buffers, as well ashydrochloric acid, sodium hydroxide, magnesium oxide, monopotassiumphosphate, bicarbonate, ammonia, carbonic acid, hydrochloric acid,sodium citrate, citric acid, acetic acid, disodium hydrogen phosphate,borax, boric acid, sodium hydroxide, diethyl barbituric acid, andproteins, as well as various biological buffers, for example, TAPS,Bicine, Tris, Tricine, HEPES, TES, MOPS, PIPES, cacodylate, or MES.

In various embodiments described herein, the ionic strength modifyingagents include those agents known in the art, for example, glycerin,propylene glycol, mannitol, glucose, dextrose, sorbitol, sodiumchloride, potassium chloride, and other electrolytes.

Useful viscosity modulating agents include but are not limited to, ionicand non-ionic water soluble polymers; crosslinked acrylic acid polymerssuch as the “carbomer” family of polymers, e.g., carboxypolyalkylenesthat may be obtained commercially under the Carbopol® trademark;hydrophilic polymers such as polyethylene oxides,polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol;cellulosic polymers and cellulosic polymer derivatives such ashydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethylcellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethyl cellulose, and etherified cellulose; gums suchas tragacanth and xanthan gum; sodium alginate; gelatin, hyaluronic acidand salts thereof, chitosans, gellans or any combination thereof.Typically, non-acidic viscosity enhancing agents, such as a neutral orbasic agent are employed in order to facilitate achieving the desired pHof the formulation.

In various embodiments described herein, formulations for injection maybe suitably formulated as a sterile non-aqueous solution or as a driedform (e.g., lyophilized) to be used in conjunction with a suitablevehicle such as sterile, pyrogen-free water. The preparation offormulations for injection under sterile conditions, for example, bylyophilisation, may readily be accomplished using standardpharmaceutical techniques well-known to those skilled in the art. In oneembodiment, the viscosity of a solution containing hyaluronic acid isincreased by addition of a hyaluronic acid-binding syntheticpeptidoglycan.

In various embodiments described herein, the solubility of a hyaluronicacid-binding synthetic peptidoglycan used in the preparation offormulations for administration via injection may be increased by theuse of appropriate formulation techniques, such as the incorporation ofsolubility-enhancing compositions such as mannitol, ethanol, glycerin,polyethylene glycols, propylene glycol, poloxomers, and others known tothose of skill in the art.

In various embodiments described herein, formulations for administrationvia injection may be formulated to be for immediate and/or modifiedrelease. Modified release formulations include delayed, sustained,pulsed, controlled, targeted and programmed release formulations. Thus,a hyaluronic acid-binding synthetic peptidoglycan may be formulated as asolid, semi-solid, or thixotropic liquid for administration as animplanted depot providing modified release of the active compound.Illustrative examples of such formulations include drug-coated stentsand copolymeric(dl-lactic, glycolic)acid (PGLA) microspheres. In anotherembodiment, hyaluronic acid-binding synthetic peptidoglycans orcompositions comprising hyaluronic acid-binding synthetic peptidoglycanmay be continuously administered, where appropriate.

In any of the embodiments described herein, the hyaluronic acid-bindingsynthetic peptidoglycan can be administered alone or in combination withsuitable pharmaceutical carriers or diluents. Diluent or carrieringredients used in the hyaluronic acid-binding synthetic peptidoglycanformulation can be selected so that they do not diminish the desiredeffects of the hyaluronic acid-binding synthetic peptidoglycan. Thehyaluronic acid- binding synthetic peptidoglycan formulation may be inany suitable form. Examples of suitable dosage forms include aqueoussolutions of the hyaluronic acid-binding synthetic peptidoglycan, forexample, a solution in isotonic saline, 5% glucose or other well-knownpharmaceutically acceptable liquid carriers such as alcohols, glycols,esters and amides.

Suitable dosages of the hyaluronic acid-binding synthetic peptidoglycancan be determined by standard methods, for example by establishingdose-response curves in laboratory animal models or in clinical trials.In various embodiments described herein, the dosage of the hyaluronicacid-binding synthetic peptidoglycan, can vary significantly dependingon the patient condition, the disease state being treated, the route ofadministration and tissue distribution, and the possibility of co-usageof other therapeutic treatments. Illustratively, suitable dosages ofhyaluronic acid-binding synthetic peptidoglycan (administered in asingle bolus or over time) include from about 1 ng/kg to about 10 mg/kg,from about 100 ng/kg to about 1 mg/kg, from about 1 μg/kg to about 500μg/kg, or from about 100 μg/kg to about 400 μg/kg. In each of theseembodiments, dose/kg refers to the dose per kilogram of patient mass orbody weight. In other illustrative aspects, effective doses can rangefrom about 0.01 μg to about 1000 mg per dose, from about 1 μg to about100 mg per dose, or from about 100 μg to about 50 mg per dose, or fromabout 500 μg to about 10 mg per dose, or from about 1 mg to 10 mg perdose, or from about 1 to about 100 mg per dose, or from about 1 mg to5000 mg per dose, or from about 1 mg to 3000 mg per dose, or from about100 mg to 3000 mg per dose, or from about 1000 mg to 3000 mg per dose.In one embodiment, suitable dosages of a hyaluronic acid-bindingsynthetic peptidoglycan include concentrations ranging from about 0.01uM to about 100 uM, about 0.05 to about 100 uM, about 0.1 uM to about100 uM, about 0.1 uM to about 50 uM, about 0.1 uM to about 20 uM, about0.1 uM to about 10 uM, about 0.5 uM to about 10 uM, about 0.5 uM toabout 50 uM, and about 0.5 uM to about 100 uM. In another embodiment,suitable dosages of a hyaluronic acid-binding synthetic peptidoglycaninclude concentrations of about 0.01 uM, 0.1 uM, 0.2 uM, 0.5 uM, 1 uM, 2uM, 5 uM, 10 uM, 20 uM, 50 uM, and 100 uM.

The hyaluronic acid-binding synthetic peptidoglycan can be formulated inan excipient. In any of the embodiments described herein, the excipientcan have a concentration ranging from about 0.4 mg/ml to about 6 mg/ml.In various embodiments, the concentration of the excipient may rangefrom about 0.5 mg/ml to about 10 mg/ml, from about 0.1 mg/ml to about 6mg/ml, from about 0.5 mg/ml to about 3 mg/ml, from about 1 mg/ml toabout 3 mg/ml, from about 0.01 mg/ml to about 10 mg/ml, and from about 2mg/ml to about 4 mg/ml.

In embodiments where the hyaluronic acid-binding synthetic peptidoglycanis implanted as part of a cartilage repair composition or device (e.g.,a gel for implantation), any suitable formulation described above may beused.

Any effective regimen for administering the hyaluronic acid-bindingsynthetic peptidoglycan can be used. For example, the hyaluronicacid-binding synthetic peptidoglycan can be administered as a singledose, or as a multiple-dose daily regimen. Further, a staggered regimen,for example, one to five days per week can be used as an alternative todaily treatment.

In various embodiments described herein, the patient is treated withmultiple injections of the hyaluronic acid-binding syntheticpeptidoglycan. In one embodiment, the patient is injected multiple times(e.g., about 2 up to about 50 times) with the hyaluronic acid- bindingsynthetic peptidoglycan, for example, at 12-72 hour intervals or at48-72 hour intervals. Additional injections of the hyaluronicacid-binding synthetic peptidoglycan can be administered to the patientat an interval of days or months after the initial injections(s).

In any of the embodiments herein described, it is to be understood thata combination of two or more hyaluronic acid-binding syntheticpeptidoglycans, differing in the peptide portion, the glycan portion, orboth, can be used in place of a single hyaluronic acid-binding syntheticpeptidoglycan.

It is also appreciated that in the foregoing embodiments, certainaspects of the compounds, compositions and methods are presented in thealternative in lists, such as, illustratively, selections for any one ormore of G and P. It is therefore to be understood that various alternateembodiments of the invention include individual members of those lists,as well as the various subsets of those lists. Each of thosecombinations is to be understood to be described herein by way of thelists.

In the following illustrative examples, the terms “aggrecan mimetic” and“mimetic” are used synonymously with the term “hyaluronic acid-bindingsynthetic peptidoglycan.”

EXAMPLE 1 Peptide Synthesis

All peptides were synthesized using a Symphony peptide synthesizer(Protein Technologies, Tucson, Ariz.), utilizing an FMOC protocol on aKnorr resin. The crude peptide was released from the resin with TFA andpurified by reverse phase chromatography on an AKTAexplorer (GEHealthcare, Piscataway, N.J.) utilizing a Grace-Vydac 218TP C-18 reversephase column and a gradient of water/acetonitrile 0.1%TFA.Dansyl-modified peptides were prepared by adding an additional couplingstep with dansyl-Gly (Sigma) before release from the resin. Peptidestructures were confirmed by mass spectrometry. The following peptideswere prepared as described above: GAHWQFNALTVRGGGC, KQKIKHVVKLKGC, andKLKSQLVKRKGC.

EXAMPLE 2 Chondroitin Sulfate Functionalization and SyntheticPeptidoglycan Formation

The reaction schematic for the creation of the aggrecan mimic (i.e.,GAH) can be seen in FIG. 1. Functionalization of the chondroitin sulfate(CS) (Sigma, St. Louis, Mo.) was accomplished using sodium periodate(Thermo Scientific, Waltham, Mass.) to oxidize the CS. By varying thereaction duration and sodium periodate concentration, the number ofaldehyde groups produced by the oxidation reaction was controlled,values presented in Table 2. Table 2 details the sodium periodateconcentration and the reaction duration needed to obtain the desirednumber of aldehydes per CS chain. Through progressive chemicalreactions, schematic shown in FIG. 1, the number of BMPH attached per CSchain is assumed to equal the number of aldehydes produced and thenumber of hyaluronic acid (HA) binding peptides attached.

Based on the reaction duration and the concentration of sodiumperiodate, the number of peptides (average) per CS chain is shown inTable 2.

TABLE 2 Sodium Periodate Reaction Concentration Duration # Aldehydes/(mM) (hr) CS Chain 10 24 3 20 24 7.2 30 24 8.5 20 48 9 30 48 10.5

The concentration of CS was kept constant at 20 mg per mL for alloxidation reactions. The measured amounts of CS and sodium periodatewere reacted and protected from light in 0.1 M sodium acetate buffer (pH5.5) for the durations specified. Completion of the reaction wasobtained by removing sodium periodate by performing gel filtrationchromatography with a Bio-Scale Mini Bio-Gel column packed withpolyacrylamide beads (Bio-Rad Laboratories, Hercules, Calif.) using anÄKTA Purifier FPLC (GE Healthcare, Piscataway, N.J.). The running bufferused for the desalting process was 1× Phosphate Buffered Saline (PBS, pH7.4, Invitrogen, Carlsbad, Calif.).

N-[β-Maleimidopropionic acid]hydrazide,trifluoroacetic acid salt (BMPH,Pierce, Rockford, Ill.) was reacted in a 50 M excess with the desalted,oxidized CS in 1× PBS. The hydrazide end of BMPH reacts to covalentlyattach to the functionalized CS, via the newly created aldehydes, toform a Schiff base intermediate. Sodium cyanoborohydride (5 M, Pierce)was added to the reaction to reduce the Schiff base intermediate imineto a more stable amine. Excess BMPH was removed from the solution byFPLC desalting in deionized water. Due to the absorbance detectioncapabilities on the ÄKTA Purifier FPLC, the amount of excess BMPH wasmeasured. The small size and low molecular weight of BMPH (297.19 g/mol)resulted in its elution from the column at a separate, much latertimepoint. With the presence of its numerous single bonds and occasionaldouble bonds, BMPH produced a strong absorbance spectrum at both the 215nm wavelength (characteristic of single bonds) and 254 nm wavelength(characteristic of double bonds). Therefore, a standard curve wasproduced, correlating known BMPH masses to the integrated area of the215 nm absorbance spectra, FIG. 2. With this standard curve, the mass ofexcess BMPH was determined. Subtracting the excess BMPH mass from theoriginal reaction mass allows the determination of the mass of BMPHconsumed in the reaction. Using the consumed mass, the number of BMPHbound to the oxidized CS was calculated. The collected CS-BMPH productwas frozen, lyophilized, and stored at −80° Celsius.

The HA binding peptide sequence was identified by Mummert. Slightmodifications to the identified sequence produced the specific HAbinding sequence, GAHWQFNALTVRGGGC (noted as GAH), that was used in thisresearch. The peptide was produced by and purchased from Genscript(Piscataway, N.J.). The cysteine amino acid was included to allowcoupling, by way of thioether bond formation, to the maleimide group ofBMPH. This reaction occurs at a 1:1 ratio, allowing the assumption thatthe number of BMPH bound to the functionalized CS will equal the numberof GAH peptides attached. GAH peptide, at one molar excess to the numberof BMPH coupled per chain, was dissolved in dimethyl sulfoxide (DMSO,Sigma) and was added to the CS-BMPH solution in 15 minute intervals, aquarter of the volume at a time. After the last addition of GAH peptide,the reaction was allowed to progress for two hours. During this time,the excess GAH peptide formed particulates. Before purifying thesolution to obtain GAH functionalized CS, the solution was passedthrough an Acrodisc 0.8 μm pore diameter filter (Pall, Port Washington,N.Y.) to remove the excess peptide particulates. The solution was thenpassed, with deionized water, through the ÄKTA Purifier FPLC to purifythe GAH-CS compound. The collected compounds were then frozen at −80°Celsius and lyophilized to produce the desired aggrecan-mimics. Bylaboratory convention, the aggrecan mimic was named by (# of peptidesattached) (first three letters of peptide sequence)—(GAG abbreviationthat was functionalized) i.e. for the aggrecan mimic, 3GAH-CS for 3 GAHHA binding peptides functionalized to a chondroitin sulfate GAGbackbone.

EXAMPLE 3 Binding of Synthetic Peptidoglycan to Hyaluronic AcidSynthetic Peptidoglycan Binding to Immobilized Hyaluronic Acid

Hyaluronic Acid (HA, from Streptococcus equi, Sigma) at a concentrationof 4 mg per mL, was immobilized to a 96-well plate (Costar, blk/clr,Corning, Corning, N.Y.) overnight at 4° Celsius. Biotin labeled GAHpeptides were bound, by way of BMPH, to functionalized CS at aconcentration of 1 biotin-GAH per CS chain. Unlabeled GAH peptides boundto the remaining unreacted aldehydes of CS. Standard biotin-streptavidindetection methods were utilized to determine the degree of aggrecanmimic binding to the immobilized HA. Blocking of the HA surface was donefor one hour with 1% Bovine Serum Album (BSA, Sera Care Life Sciences,Milford, Mass.) in 1× PBS solution. After washing with 1× PBS, thebiotin-labeled aggrecan mimic was incubated in the well for 30 minutesand then washed with 1× PBS. Streptavidin-horseradish peroxidase (R&DSystems, Minneapolis, Minn.) solution was added to each well, andallowed to react for 20 minutes. After reaction completion and washing,chromogen solution was added (Substrate Reagent Pack, R&D Systems) anddeveloped for 15 min. At 15 min, sulfuric acid (Sigma) was addeddirectly to each well to stop the reaction. The well plate was then readon the M5 SpectraMax Plate Reader (Molecular Devices, Sunnyvale, Calif.)at 450 and 540 nm wavelengths. By subtracting the two absorbancereadings produced, the absorbance due to the bound biotin-labeledaggrecan-mimic was determined.

One GAH peptide per aggrecan mimic was replaced by a biotin-labeled GAHpeptide and the now-labeled aggrecan mimic was incubated withimmobilized HA. Commercially available biotin detection products(through streptavidin and HRP) demonstrated the degree of mimic bindingto the immobilized HA (see FIG. 3). Starting at a concentration of 1 μM,the aggrecan mimic had a dose dependent increase in presence on theimmobilized HA, proving that the mimic was binding to the HA. However,the determination of the mimic's binding affinity was not pursued due tothe uncertainty of the amount of HA immobilized.

Rheometer Derived Synthetic Peptidoglycan Binding to Hyaluronic Acid

HA solutions were created to test the aggrecan-mimic's ability to bindto HA in a more physiologically relevant situation. The ability of theaggrecan-mimic to bind to HA was deduced by the improvement in storagemodulus of the solution, indicating HA crosslinking by the mimic.Multiple treatments were created in 1× PBS pH 7.4 to test the aggrecanmimic's ability to bind HA: 2.5 wt % HA control, HA+CS at a 25:1 molarratio of CS:HA, HA+3GAH-CS at 25:1, HA+7.2GAH-CS at 25:1, HA+10.5GAH-CSat 25:1.

Using the AR-G2 Rheometer (TA Instruments, New Castle, Del.), frequency(0.1-100 Hz, 2.512 Pa) and stress (0.1-100 Pa, 1.0 Hz) sweeps wereconducted to measure the storage modulus of each solution.

Rheology studies the flow of a substance in response to applied forcesand is often used when measuring viscoelastic materials. In particular,the rheometer determines the storage modulus and the loss modulus basedon the substance feedback to the applied force. The storage modulus is ameasure of the amount of energy that is elastically absorbed by thesubstance and the loss modulus depicts the amount of energy lost throughheat. A large storage modulus is indicative of a gel-like substance witha more rigid, elastic structure; whereas, a small storage modulus and alarge loss modulus indicate a viscous material that does not elasticallyretain the applied load. The high molecular weight HA (˜1 .5 MDa) is avery viscous material which elastically retains a portion of the appliedload due to a pseudo-gel formed by HA chain entanglement. The createdaggrecan mimic contains multiple HA binding peptides which can act as atype of HA chain crosslinker assuming adequate mimic binding to the HA.In solution with the high molecular weight HA, it is hypothesized thatthe aggrecan mimic could increase the rigidity of the solution, creatinga larger storage modulus. A larger storage modulus would be indicativeof extensive HA crosslinking, proving a strong binding affinity betweenthe aggrecan mimic and the HA chains present in the mixture. Multipleversions of the aggrecan mimic were tested, differentiated by the numberof GAH peptides (on average either 3, 7.2, or 10.5) attached perfunctionalized CS chain.

The results of the experiment, shown in FIG. 4, showed that the additionof CS significantly (α=0.05) lowered the storage modulus of the HAsolution. The addition of the dense negative charges associated with theCS helped spread the HA chains, easing the degree of HA entanglement andremoving the pseudo-gel that stored the applied energy. Confirming thehypothesis, as the number of GAH peptides per CS increased from 3 to10.5, the storage modulus of the mixture increased as well. Thisincrease can be attributed to two beneficial attributes of having ahigher number of GAH peptides per aggrecan mimic. First, the more GAHpeptides attached per CS, the higher the avidity of the mimic, resultingin a stronger mimic binding to the HA molecule. Second, the more GAHpeptides attached per CS, the greater the likelihood of the mimic actingas a crosslinker between the HA molecules. Both effects contributed to amore gel-like mixture, resulting in a larger measured storage modulus.Weaker binding between the mimic and HA would not restore the pseudo-geland would be unable to store the applied energy from the rheometer. Theincrease in storage modulus confirms the strong mimic binding to theimmobilized HA shown in FIG. 3. Specifically at 10.5 GAH peptides per CSchain, the storage modulus was significantly (α=0.05) higher than theHA+CS control, reaching an average storage modulus similar to the HAcontrol.

EXAMPLE 4 Synthetic Peptidoglycan Compression Studies Collagen GelFormation and Turbidity

To mimic the native cartilage extracellular matrix, collagen wasutilized to entrap the HA and aggrecan-mimic aggregates within a naturalscaffold. Collagen type II (CII) was obtained from two differentcommercial sources (Affymetrix, Santa Clara, Calif. and Sigma). Mixturesof the cartilage ECM components were prepared in TES Buffer (60 mM TES,20 mM Na₂HPO₄, 0.56 M NaCl, chemicals from Sigma) pH 7.6 according tothe native component breakdown, where CII comprised 70 dry wt % and thecombination of HA and the aggrecan mimic/CS control formed the remaining30 dry wt % of the mixture. The final concentration of CII in the gelwas 2 mg per ml. Samples consisted of a CII control, CII+HA+CS control,and CII+HA+aggrecan mimic (10.5 GAH-CS). To prevent prematurefibrillogenesis and gel formation, the solutions were kept on ice at anacidic pH. Solution mixtures of the components were placed in a 384 wellplate (Greinier blk/clr, Monroe, N.C.), placed at 37° C. andphysiological pH to initiate fibrillogenesis, and were monitored at 313nm on the M5 SpectraMax to determine gel formation. CII was unable toform gels when included with the varying treatments (See SupplementaryInformation). Therefore, collagen type I (CI, High Concentration RatTail Collagen Type 1, BD Biosciences, Bedford, Mass.) was utilized forthe gel formation. The same treatments and procedure were used with theCI, except that the component masses were shifted for a CI finalconcentration of 4 mg per mL. CI was used for all following experiments.

Turbidity with CI was performed to measure the formation of thecartilage replicate, results shown in FIG. 5. As demonstrated, theaddition of HA+10.5 GAH-CS did not affect the fibrillogenesis of thecollagen fibers. All treatments followed a similar curve and reachedsimilar absorbance peaks at about the same time. HA+10.5 GAH-CStreatment had a higher initial absorbance due to the aggrecan mimicstendency to form self-aggregates in 1× PBS solution, not due topremature CI fibril formation. The aggregation of 10.5 GAH-CS wasrecognized during the initial HA rheometer tests, but the aggregationdid not inhibit the aggrecan mimic's ability to bind to HA.

Collagen Gel Property Testing

Collagen-based gel compression tests and frequency sweeps were conductedusing an AR-G2 Rheometer using a 20-millimeter parallel plate geometry(TA Instruments). The 375 μL gel mixtures were prepared on ice andpipetted onto the rheometer base plate. The geometry was lowered to agap distance of 1 mm and the solution was heated to 37° Celsius. Ahumidity trap was utilized to prevent gel dehydration while the mixturewas allowed to gel over two hours. This two hour value was determined bythe demonstrated time to gelation data from the turbidity data. Afterthe two hour time period, the gels were compressed or oscillateddepending on the test. Compression tests occurred at an engineeringstrain rate of 1% (10 μm) per second. The gap distance and the normalforce on the geometry head were measured. The frequency sweeps measuredthe storage modulus of the created gels during a logarithmic base tenincrease in frequency from 0.1 to 1 Hz.

The simultaneous normal force and displacement were measured, and theengineering stress and strain were calculated for the treatments. Asshown in FIG. 6, the inclusion of the aggrecan mimic significantly(α=0.05) increased the compressive strength of the gel complex. The peakengineering stress of the collagen+HA+AGG mimic reached 7.5 kPa at anengineering strain of 9%, whereas the collagen+HA+CS control reached apeak of 4.8 kPa at 4%, and the collagen control reached a peak of 4.2kPa at 15% strain.

Two factors contributed to the increase in compressive strength of theCI+HA+10.5 gel, the first being the mimic's ability to attract water andthe second being the HA crosslinking ability of the aggrecan mimic. Innative cartilage, the predominance of the entrapped negative chargesprovided by the HA and CS attract water and retard its diffusion fromthe cartilage. When a compressive force is applied to the cartilage, thewater is not able to diffuse out into the synovial capsule. Retainingthis incompressible water increases the compressive strength of thestructure. Similarly in the tested gel complexes, the inclusion of thenegative charges associated with CS in the gel provides the sameattraction. As can be seen in FIG. 6, both the CS and 10.5 GAH-CStreatments have an increased compressive strength. The CS treatment isnot fixed within the CI complex (it is not bound to HA) and thereforeafter a small compressive deformation, the CS and its attracted waterdiffuse out of the complex into the surrounding fluid. The diffusion ofthe CS and water from the complex diminishes the compressive strength ofthe complex, causing the resulting gel's compressive profile to resemblethat of the collagen scaffold control. In contrast, 10.5 GAH-CS is boundto the interwoven HA. Therefore, a much higher compressive stress isrequired to overcome the binding of the mimic to HA and cause thediffusion of CS and attracted water from the complex.

Secondly, the ability of the aggrecan mimic to act as a HA crosslinkerresults in a higher degree of entrapment for the HA and mimic.Effectively, the HA crosslinking nature creates large aggregates withinthe collagen complex, similar to the native aggrecan/HA aggregates. Themain difference between the aggrecan mimic and native aggrecan is thesize of the molecule. The protein backbone of aggrecan alone weighs ˜220kDa, whereas the aggrecan mimic, in entirety, only weighs around 30 kDa.Therefore, the native aggregate complex, with over 100 aggrecanmolecules bound to the HA, produces much larger aggregates than theaggrecan mimic could produce. However, by acting as a crosslinkerbetween HA chains, the aggrecan mimic can produce its own form of anaggregate that also portrays the main characteristics of nativeaggregates; voluminous, negatively-charged structures. The role of theaggrecan mimic as an HA crosslinker was further investigated by applyingshear loads through rheo logical tests on the CI gels described above.The results of these experiments can be seen in FIG. 7.

The inclusion of 10.5 GAH-CS significantly (α=0.05) increased thestorage modulus of the formed gel. The network created by the binding ofthe mimic to the HA supplemented the existing rigidity of the CI matrix,allowing an increased elastic absorbance of the energy applied by shearloading. This study was important as it verified the crosslinkingability of the 10.5 GAH-CS and the creation of an alternate aggregateform.

EXAMPLE 5 Synthetic Peptidoglycan Protection of Hyaluronic AcidDegradation

Dynamic viscosity values of HA solutions were determined using theAR-G2. High molecular weight HA solutions have a large viscosity due tothe extensive chain entanglement caused by the long chain length.Hyaluronidase (Type II from Sheep Testes, Sigma) cleaves the HA chain,creating shorter chains with less entanglement. The shorter HA chainswill have a measurably lower viscosity. HA solutions were incubated with100 units/mL hyaluronidase. Dynamic viscosities were determined using atime sweep with constant angular frequency and oscillatory stressinitially and at 2 and 4-hour timepoints. Samples (at 0.5 wt % HA)consisted of HA, HA+CS, and HA+10.5 GAH-CS. The treatment values wereadded at a 75:1 treatment to HA molar ratio. The percent degradation wascalculated for each measurement by dividing the initial viscosity fromthe difference of the measured viscosity minus the initial viscosity.

Work by Pratta et al. and Little et al. has shown the importance ofaggrecan in preventing cartilage component degradation. The demolitionof the cartilage matrix in osteoarthritis is started with the cleavageof the aggrecan proteoglycans. The removal of the

GAG-rich region of the proteoglycan exposes the remaining components,CII and HA, to degrading enzymes. With the knowledge of the importanceof aggrecan in preventing degradation, studies were conducted todetermine the ability of the aggrecan-mimic in preventing HAdegradation.

The viscosity of a HA solution is dependent on the size of the HAchains. Due to entanglement, larger HA chains will produce a higherviscosity. When exposed to hyaluronidase, the HA chain is cleaved intosmaller units. Therefore, the size of the HA and the amount of HAentanglement decreases. This decrease prompts a similar decrease in themeasured viscosity. The percent change in viscosity of HA solutions inthe presence of hyaluronidase will provide key information into theamount of degradation the HA has undergone. FIG. 8 presents the percentdegradation of HA control versus the associated treatments. As can beseen, the AGG mimic, GAH, significantly reduced the rate of degradationof HA, indiating that it behaves similarly to native AGG in itsprotection of ECM components.

Viscosities of each treatment without hyaluronidase (TES Buffer replacedthe hyaluronidase volume) were initially measured and served as thebaseline for the percent degradation calculations. The 0 hr timepointinvolved the addition of the hyaluronidase, mixing of the solution,pipetting onto the rheometer, and the beginning equilibration operationof the machine Therefore, the 0 hr timepoint occurred approximately twominutes after the addition of hyaluronidase. A high concentration ofhyaluronidase (25 units per mL) was utilized to replicate the worstpossible scenario. In addition, the HA molecules were dispersed insolution, rather than tightly interwoven into a collagen network. As canbe seen from FIG. 8, both the HA Control and the HA+CS treatment hadalmost complete degradation of the HA solution at the 0 hr timepoint. Incontrast, the addition of 10.5 GAH-CS significantly (α=0.05) reduced theamount of HA degradation. In fact, the presence of 10.5 GAH-CS increasedthe viscosity above the baseline values. It is believed that theaddition of hyaluronidase cleaves some of the excess HA. This allows10.5GAH-CS to better crosslink the remaining, intact chains, creating adenser gel which produced the larger viscosity. At the 2 hr timepoint,both the HA control and HA+CS had completely degraded with percentdegradations above 90%, but the HA solution with 10.5 GAH-CS had asignificantly (α=0.05) lower percent degradation. Lastly, at the 4 hrtimepoint, all treatments had been degraded, with their percentdegradations all above 90%. Amongst the three timepoints, 10.5 GAH-CSwas not able to completely prevent HA degradation, but it drasticallyreduced the rate of degradation compared to the degradations of the HAControl and HA+CS. This reduced rate demonstrates that the 10.5 GAH-CSprevents the degradation of the HA chains. It is believed that thisprevention is being accomplished through competitive inhibition of thehyaluronidase cleavage point on the HA chain. The non-covalent bindingof the mimic to the HA chain coupled with the gradual degradation rateof the HA chains appear to validate this belief. In addition, thedegradation rate of the 10.5 GAH-CS solution is still believed to beartificially high. Upon incubation of the mimic within the HA solution,HA+10.5 GAH-CS aggregates were formed. However, these aggregates did notspread uniformly throughout the solution volume. Therefore, thesolutions were mixed, similarly to the other samples, before ameasurement was taken. The mixing of the solution disrupted theaggregates, dislodging 10.5 GAH-CS and exposing the hyaluronidasecleavage point. Even after the 4 hr timepoint, when supposedly completedegradation had occurred, substantial aggregation of HA+10.5 GAH-CSstill occurred. In a compact matrix like the ECM of cartilage, it ispossible that 10.5 GAH-CS could not only significantly reduce thedegradation rate, but suppress HA degradation.

EXAMPLE 6 CryoScanning Electron Microscopy (SEM)

The ECM-based constructs, as described for turbidity measurements, wereformed on an SEM plate at 37° C. overnight. The SEM plates were securedinto a holder, and were plunged into a liquid nitrogen slush. A vacuumwas pulled on the sample as it was transferred to the Gatan Alto 2500pre-chamber. Within the chamber, cooled to −170° C., a cooled scalpelwas used to create a free break surface on the sample. The sample wassubjugated to sublimation at −85° C. for 15 minutes followed by asputter-coating of platinum for 120 seconds. After sputter-coating, thesample was transferred to the microscope stage and images were taken at−130° C.

Representative images were obtained at a magnification of 10,000×, asshown in FIG. 9. Panel A shows the CI control, and is characterized byextensive crosslinking between major fibrils, and relatively smallmatrix pore size. Panel B shows CI+HA+CS, and contains extensivecrosslinking, but larger pore size, due to the presence of the large HAchains. Panel C shows CI+HA+10.5 GAH-CS and illustrates a noticeablysmaller degree of crosslinks in addition to a very large pore size. TheAGG mimic can bind to the HA creating a relatively large, cumbersomecomplex that hinders the CI crosslinking

As can be qualified in the representative images, the addition of HA+CSdid not have an effect on the variation of collagen fibril diameters,but the HA+CS sample did have a larger representative void space. Incomparison to the control groups, the addition of the AGG mimic with theHA resulted in a smaller variation of collagen fibril diameters due tothe limited number of small fibril diameters, and an overall increase inthe void space of the sample. The binding of the AGG mimic to the HAmolecule created an aggregate complex that was trapped within thecollagen scaffold and excluded smaller fibril formation between thelarger fibrils due to steric hindrance.

EXAMPLE 7 Collagen Protection

ECM-based constructs containing collagen alone, collagen+HA+CS, orcollagen+HA+10.5 GAH-CS were created in 8-well chambered slides asdescribed previously. The final sample volume was 200 μL consisting of0.8 mg of collagen type I. Matrix metalloprotease-I (MMP-I, R&D Systems,Minneapolis, Minn.) at a concentration of 0.133 mg/mL, was activatedfollowing the protocol detailed in the manufacturer's instructions.Briefly, MMP-1, already dissolved in manufacturer's buffer (50 mM Tris,10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35, pH 7.5), was combined with anequal volume of 25 mM APMA (Sigma) in DMSO at 37° C. for 2 hrs toactivate the enzyme. Upon activation, the MMP-1 solution was diluted twofold in water and was added to the sample as a 100 μL supernatant. Thesamples were incubated at 37° C. with gentle shaking Twenty-five hrsafter the addition of the initial enzyme solution, the supernatant wasremoved and replaced with a fresh batch of enzyme. After 50 total hr ofincubation with the enzyme, the remaining gels were removed from thechambered slides, washed with deionized water to remove any enzymesolution or degradation products, and resolubilized in 12 M HCl. Thesamples were diluted in water to reach a final concentration of 6 M HCl,and were hydrolyzed overnight at 110° C. Following hydrolysis, theamount of hydroxyproline (hyp) was analyzed according to the protocoldeveloped by Reddy, et al. (Clin Biochem, 1996, 29: 225-9). Briefly, thehydrolyzed samples were incubated with Cholramine T solution (0.56 M)for 25 minutes at room temperature before the addition of Elrich'sreagent and subsequent chlorophore development for 20 minutes at 65° C.After the development of the chlorophore, the samples were read on aspectrophotometer at a wavelength of 550 nm. Absorbance readings werecompared to those obtained from known concentrations of collagen todetermine the amount of collagen remaining in each sample.

Each replicate sample was constructed with 0.8 mg of CI, and afterdegradation, the remaining CI amount was determined by the protocoldeveloped by Reddy et al. and converting that to CI amount by a set ofCI standards. The percent degradation was determined by subtracting theremaining CI from the initial CI, dividing by the initial CI, andmultiplying by 100. The percent degradation of the three treatments isshown in FIG. 10. All the treatments were significantly different fromeach other (p<0.05). In particular, the percent degradation of the AGGmimic sample (CI+HA+10.5 GAH-CS =41.0%) was significantly less (p<0.05)than the other two treatments (CI=64.5% and CI+HA+CS=74.7%). Thepresence of the AGG mimic significantly reduced the CI degradation. Thepresence of the AGG mimic can act as a hindrance to the cleavage sitesof the degrading enzymes. By creating the large aggregates with HA thatare tightly trapped within the collagen scaffold, the AGG mimic canoccupy the space proximal to the collagen, preventing enzyme access todegradation locations.

EXAMPLE 8 Diffusion of Peptidoglycans Through Cartilage Matrix

Cartilage explants were obtained from the load bearing region of threemonth old bovine knee joints. Native aggrecan was removed from harvestedcartilage explants leaving a matrix consisting primarily of type IIcollagen and residual GAG. This was achieved by treating explants with0.5% (w/v) trypsin in HBSS for 3 hours at 37° C. (FIG. 13). Aftertrypsin treatment explants were washed three times in HBSS and incubatedwith 20% FBS to inactivate residual trypsin activity. Peptidoglycan wasdissolved in distilled water at 10 μM concentration and diffused throughthe articular surface of cartilage explants by placing 10 μL of thesolution on the surface every ten minutes for one hour at roomtemperature (FIG. 14). Normal cartilage and aggrecan depleted cartilagewere treated with 1× PBS as positive and negative controls respectively.After diffusion, explants were washed three times with 1× PBS and storedat −20° C. until further testing. Diffusion of peptidoglycan wasconfirmed by staining a midsagittal section of the tissue withstreptavidin-horseradish peroxidase stain. The streptavidin stain bindsto the biotin labeled molecule and is depicted as a brown color (FIGS.15 and 16).

EXAMPLE 9 Bulk Compression Testing

Displacement-controlled unconfined compression was performed on an AR G2rheometer with force transducers capable of detecting normal forces inthe range of 0.01-50 N (TA Instruments). The explants were glued to thebottom of a hydrophobic printed slide

(Tekdon) and covered in a 1× PBS bath. A 20 mm diameter stainless steelparallel plate geometry head was lowered until initial contact was made.Explant height was measured using a digital micrometer (Duratool).Compressive loads from 0-30% nominal strain (at 5% intervals) wereapplied to the explants through a stepwise loading that involved a rampduration of 5 sec (i.e. a strain rate of 1.0%/sec) and hold time of 30sec. Compressive stiffness values were obtained by using the slope ofequilibrium stress values, computed during each hold section, versusrespective strain values, based on a linear fit model. Scaffolds testedfor bulk compression included: 1) Normal cartilage, 2) Aggrccan depletedcartilage (AD), and 3) AD+mAGC (FIG. 17). Addition of the HA bindingpeptidoglycan (mAGC) significantly restored stiffness of cartilageexplants to a higher extent as compared to the collagen type II bindingpeptidoglycan (mAG(II)C).

EXAMPLE 10 Animal Model

Sprague-Dawley rats (250-300g) were used for surgery. The patellartendon, the anterior and posterior cruciate ligaments and the medial,lateral collateral ligaments were transected. The medial and lateralmeniscuses were totally menisectomized. The knee joint capsule wasrepaired with an absorbable suture and the skin was closed with a 4-0monofilament nylon. Starting at week 4, 10 μl of a 1 μm mAGC wasadministered weekly.

The extent of inflammation was indicated by the MMP-13 probe (FIG. 18)in Sprague-Dawley rats treated with and without peptidoglycan at four,six and eight weeks post surgery (FIG. 19). X-ray images ofSprague-Dawley rat knee joints showed injured knee 6 weeks and 8 weeksfollowing OA induction (FIG. 20, Panels A and D, respectively), injuredknee with peptidoglycan treatment (FIG. 20, Panels B and E,respectively), and normal knee (FIG. 20, Panel C) six weeks afterosteoarthritis induction surgery. MicroCT of Sprague-Dawley ratsindicated re-growth of new cartilage six and eight weeks after OAinduction surgery. Injured knees 6 weeks and 8 weeks following OAinduction, (FIG. 21, Panels A and D, respectively), injured kneesfollowing peptidoglycan treatment (FIG. 21, Panels B and E,respectively), and Normal knee (FIG. 21, Panel C), are shown.

EXAMPLE 11 Reagents

Peptide GAHWQFNALTVRGGGC (GAH) was purchased from Genscript (Piscataway,N.J.). N-[β-maleimidopropionic acid] hydrazide, trifluoroacetic acidsalt (BMPH) was purchased from Pierce (Rockford, Ill.). Rat tail type Icollagen was purchased from BD Biosciences (Bedford, Mass.). Humanrecombinant interlukin-1β was purchased from Peprotech (Rocky Hill,N.J.). All other supplies were purchased from VWR (West Chester, Pa.) orSigma-Aldrich (St. Louis, Mo.) unless otherwise noted.

EXAMPLE 12 Collagen Scaffold Synthesis

Collagen scaffolds were prepared in TES buffer (60 mM TES, 20 mM Na₂PO₄,0.56 M NaCl) at a pH of 7.6. Scaffold composition for mechanical testingand in vitro inflammatory model studies are described in theirrespective sections. All solutions were maintained on ice untilfibrillogenesis was initiated at 37° C. Aligned collagen scaffolds werecreated by placing the collagen solution at the isocenter of a 9.4 Teslamagnet (Chemagnetics CMX400) at 37° C. for one hour, whereas unalignedgels were prepared similarly but without magnetic exposure. The slidecontaining the collagen solution was placed parallel to the magneticfield, orienting the collagen fibers in a direction perpendicular to thebottom of the slide. The gels were then maintained at 37° C. for 24hours in a humidity-controlled chamber to prevent evaporation.

EXAMPLE 13 Rheological Mechanical Testing

Shear and compression testing was performed on a stress-controlled AR G2rheometer (TA Instruments) using a 20 mm diameter stainless steelparallel plate geometry head. Collagen scaffolds were prepared on 20 mmdiameter hydrophobic printed slides (Tekdon). For shear tests, thegeometry head was lowered until contact was made at a gap height of 950μm. Preliminary frequency and stress sweeps were performed to determinea linear and stress-independent storage modulus range. Frequency sweepswere then performed on all gels with an oscillatory stress of 0.2 Paover a frequency range of 0.1 to 2 Hz. For compression tests, thegeometry head was lowered until contact was made with the scaffold at agap height of 1000 μm. Compressive loads from 0-30% nominal strain (at5% intervals) were applied to the collagen scaffold through a stepwiseloading that involved a ramp duration of 5 sec (i.e. a strain rate of1.0%/sec) and hold time of 30 sec. Compressive stiffness values wereobtained by using the slope of equilibrium stress values, computedduring each hold section, versus respective strain values, based on alinear fit model. Collagen scaffold composition for mechanical testswere: 1) Unaligned collagen, 2) Aligned collagen, 3) Unalignedcollagen+mAGC and 4) Aligned collagen+mAGC.

Bulk Mechanical Analysis: The aggrecan mimic, mAGC, enhanced bulkmechanical properties of scaffolds, irrespective of fiber alignment(FIG. 22). For shear testing, the storage moduli values at 0.5 Hz forunaligned and aligned collagen gels were 104.1±3.6 Pa and 49.9±5.4 Parespectively. The addition of mAGC to the collagen scaffold showed asignificant increase in the storage moduli of the unaligned and alignedgels to 113.9±4.6 Pa and 76.6±3.6 Pa respectively (p<0.001). Unalignedgels showed a higher storage modulus as compared to aligned gels(p<0.0001). For compression testing, the compressive stiffness foraligned scaffolds (2478±250 Pa) was lower than unaligned scaffolds(3564±315 Pa) (p<0.001). Addition of mAGC to these scaffold systemsincreased compressive stiffness of the aligned and unaligned scaffoldsto 4626±385 Pa and 5747±306 Pa, respectively (p<0.0001).

EXAMPLE 14 In Vitro Inflammation Model

Collagen scaffolds seeded with chondrocytes were stimulated with IL-1βand assessed for degradation products.

Chondrocyte Isolation: Primary chondrocytes were harvested fromthree-month-old bovine knee joints obtained from an abattoir within 24hours of slaughter (Dutch Valley Veal). Cartilage slices, 150-200 μmthick, were shaved from the lateral femoral condyle and washed threetimes in serum-free DMEM/F-12 medium (50 μg/mL ascorbic acid2-phosphate, 100 μg/mL sodium pyruvate, 0.1% bovine serum albumin, 100units/mL penicillin, 100 μg/mL streptomycin and 25 mM HEPES) prior todigestion with 3% fetal bovine serum (FBS) and 0.2% collagenase-P (RochePharmaceuticals) at 37° C. for six hours. Released chondrocytes werefiltered through 70 μm cell strainer and centrifuged at 1000 rpm threetimes for five minutes each in medium listed above supplemented with 10%FBS. The cell pellet was resuspended in 10% FBS supplemented media andplated on 10 cm dishes at 10,000 cells/mL in a 37° C., 5% CO₂ humidifiedincubator until confluent.

Scaffold Fabrication: Upon reaching confluency, cells were trypsinizedand encapsulated at 10,000 cells/mL within collagen scaffolds (Table 3)and allowed to equilibrate for 3 days prior to treatment.

TABLE 3 Scaffold composition for in vitro testing Unaligned CollagenExperimental Setup A: Collagen + CS + HA + IL-1β B: Collagen + CS + HAC: Collagen + mAGC + HA + IL-1β D: Collagen + mAGC + HA Aligned CollagenExperimental Setup E: Collagen + CS + HA + IL-1β F: Collagen + CS + HAG: Collagen + mAGC + HA + IL-1β H: Collagen + mAGC + HA

Inflammation Model: Constructs were incubated with or without 20 ng/mLIL-1β in chemically-defined media supplemented with 5% FBS andantibiotics (100 units/mL penicillin and 100 μg/mL streptomycin).Culture medium was replaced every two days. Removed media extracts werestored at −80° C. until further testing.

Degradation Assay: GAG degradation was monitored by measuring CSreleased in cell culture media using the dimethylmethylene blue (DMMB)dye assay and computed with a chondroitin-6-sulfate standard curve.Similarly, type I collagen degradation in cell culture media wasmonitored using the Sircol Collagen Assay using manufacturer specifiedprotocols (Bio-Color). GAG and collagen degradation were reported ascumulative release over an eight-day culture period.

Proteolytic Degradation Analysis: The amount of CS and collagen releasedinto cell culture media was significantly decreased when scaffolds thatcontained mAGC (FIGS. 11, 12, 23 and 24) (p_(cs)<0.001 andp_(collagen)<0.02, respectively). Aligned collagen gels showed astatistically higher CS and collagen release into the media as comparedto unaligned collagen fibers (p<0.001).

As described herein, the hyaluronic-binding synthetic peptidoglycan isable to protect HA and the underlying collagen fibers in the scaffoldfrom proteolytic cleavage. The synthesis of the hyaluronic-bindingsynthetic peptidoglycan utilized the chondroprotective benefits of CS.CS has been shown to down-regulate matrix metalloproteases production.Our synthetic peptidoglycan design herein described allowed CS chains tobe attached to HA, preventing degradation of both molecules. By placingthe synthetic peptidoglycan in an environment rich in proteolyticenzymes, its ability to prevent excessive loss of ECM components hasbeen demonstrated.

EXAMPLE 15 Real-time PCR

Following the cell culture study, constructs were stored in RNAlatersolution (Ambion) at 4° C. for less than one week. Total mRNA wasextracted using Nucleospin RNA II (Clontech) according to manufacturer'sprotocols. Extracted mRNA from all samples was quantified using Nanodrop2000 spectrophotometer (Thermo Scientific) and reverse transcribed intocDNA using High Capacity cDNA Reverse Transcriptase Kit (AppliedBiosystems). Real-time PCR was performed using Taqman Gene ExpressionAssays (Applied Biosystems) with the following primers: GAPDH(Bt03210913_gl), aggrecan (Bt03212186_ml) and collagen type II(Bt03251861_ml). 60 ng of cDNA template was prepared per 20 μL reactionfor the two genes of interest and the endogenous gene. Real-time PCRanalysis was carried out using a Taqman PCR Master Mix and 7500Real-Time PCR System (Applied Biosystems). Data reported was normalizedto GAPDH gene expression.

mRNA Expression Analysis: Collagen alignment, presence of aggrecan mimicand stimulation with IL-1β significantly effected aggrecan(p_(alignment)<0.001, p_(peptidoglycan)<0.02 and p_(IL-1β)<0.001) andcollagen type II expression (p_(alignment)<0.01, p_(peptidoglycan)<0.001and p_(IL-1β)<0.015). The presence of mAGC limited excessive loss of CSfrom the scaffold, which results in a lower aggrecan expression (p<0.02)(FIG. 25). The presence of mAGC also limited collagen degradation.However, collagen type II expression depended on the extent of collagenlost during degradation (FIG. 25). In unaligned scaffolds, the level ofcollagen type II expression was higher in scaffolds prepared withoutmAGC, whereas in aligned collagen scaffolds, the level of collagen typeII was higher in scaffolds prepared with mAGC (p<0.05).

EXAMPLE 16 Statistical Analysis

Each experiment was repeated twice, with at least n=3 in each data set.Statistical significance for mechanical test data was analyzed with atwo-way ANOVA with alignment and addition of peptidoglycan as factors.The cell culture data was analyzed using a three-way ANOVA withalignment, addition of peptidoglycan, and treatment with IL-1β asfactors. A post-hoc Tukey pairwise comparison (α=0.05) was used todirectly compare scaffolds prepared with and without the aggrecan mimicin each system.

1. A synthetic peptidoglycan comprising a glycan and 1 to 20 syntheticpeptides conjugated to the glycan, wherein each synthetic peptide is 5to 40 amino acids in length and comprises a hyaluronic acid-bindingamino acid sequence and wherein the synthetic peptidoglycan in thepeptidoglycan can bind to a hyaluronic acid.
 2. The syntheticpeptidoglycan of claim 1 wherein each synthetic peptide comprises anamino acid sequence of the formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2,wherein X8 is present or is not present, wherein B1 is a basic aminoacid, wherein B2 is a basic amino acid, and wherein X1-X8 are non-acidicamino acids.
 3. The synthetic peptidoglycan of claim 1 wherein eachsynthetic peptide comprises: (i) an amino acid sequence selected fromthe group consisting of: (SEQ ID NO: 2) GAHWQFNALTVRGG; (SEQ ID NO: 3)GDRRRRRMWHRQ; (SEQ ID NO: 4) GKHLGGKHRRSR; (SEQ ID NO: 5) RGTHHAQKRRS;(SEQ ID NO: 6) RRHKSGHIQGSK; (SEQ ID NO: 7) SRMHGRVRGRHE; (SEQ ID NO: 8)RRRAGLTAGRPR; (SEQ ID NO: 9) RYGGHRTSRKWV; (SEQ ID NO: 10) RSARYGHRRGVG;(SEQ ID NO: 11) GLRGNRRVFARP; (SEQ ID NO: 12) SRGQRGRLGKTR;(SEQ ID NO: 13) DRRGRSSLPKLAGPVEFPDRKIKGRR; (SEQ ID NO: 14)RMRRKGRVKHWG; (SEQ ID NO: 15) RGGARGRHKTGR; (SEQ ID NO: 16)TGARQRGLQGGWGPRHLRGKDQPPGR; (SEQ ID NO: 17) RQRRRDLTRVEG;(SEQ ID NO: 18) STKDHNRGRRNVGPVSRSTLRDPIRR; (SEQ ID NO: 19)RRIGHQVGGRRN; (SEQ ID NO: 20) RLESRAAGQRRA; (SEQ ID NO: 21)GGPRRHLGRRGH; (SEQ ID NO: 22) VSKRGHRRTAHE; (SEQ ID NO: 23) RGTRSGSTR;(SEQ ID NO: 24) RRRKKIQGRSKR; (SEQ ID NO: 25) RKSYGKYQGR;(SEQ ID NO: 26) KNGRYSISR; (SEQ ID NO: 27) RRRCGQKKK; (SEQ ID NO: 28)KQKIKHVVKLK; (SEQ ID NO: 29) KLKSQLVKRK; (SEQ ID NO: 30) RYPISRPRKR;(SEQ ID NO: 31) KVGKSPPVR; (SEQ ID NO: 32) KTFGKMKPR; (SEQ ID NO: 33)RIKWSRVSK;  and (SEQ ID NO: 34) KRTMRPTRR,

or (ii) an amino acid sequence having at least 90% sequence identity toan amino acid sequence of (i).
 4. The synthetic peptidoglycan of claim1, wherein the glycan is selected from the group consisting of dextran,chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan,heparin, keratin, and keratan sulfate. 5.-13. (canceled)
 14. A method oftreatment for arthritis in a patient, said method comprisingadministering to the patient an effective amount of the syntheticpeptidoglycan of claim
 1. 15. The method of claim 14 wherein eachsynthetic peptide comprises an amino acid sequence of the formulaB1-X1-X2-X3-X4-X5-X6-X7-X8-B2, wherein X8 is present or is not present,wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, andwherein X1-X8 are non-acidic amino acids.
 16. The method of claim 14wherein each synthetic peptide comprises: (i) an amino acid sequenceselected from the group consisting of: (SEQ ID NO: 2) GAHWQFNALTVRGG;(SEQ ID NO: 3) GDRRRRRMWHRQ; (SEQ ID NO: 4) GKHLGGKHRRSR; (SEQ ID NO: 5)RGTHHAQKRRS; (SEQ ID NO: 6) RRHKSGHIQGSK; (SEQ ID NO: 7) SRMHGRVRGRHE;(SEQ ID NO: 8) RRRAGLTAGRPR; (SEQ ID NO: 9) RYGGHRTSRKWV;(SEQ ID NO: 10) RSARYGHRRGVG; (SEQ ID NO: 11) GLRGNRRVFARP;(SEQ ID NO: 12) SRGQRGRLGKTR; (SEQ ID NO: 13)DRRGRSSLPKLAGPVEFPDRKIKGRR; (SEQ ID NO: 14) RMRRKGRVKHWG;(SEQ ID NO: 15) RGGARGRHKTGR; (SEQ ID NO: 16)TGARQRGLQGGWGPRHLRGKDQPPGR; (SEQ ID NO: 17) RQRRRDLTRVEG;(SEQ ID NO: 18) STKDHNRGRRNVGPVSRSTLRDPIRR; (SEQ ID NO: 19)RRIGHQVGGRRN; (SEQ ID NO: 20) RLESRAAGQRRA; (SEQ ID NO: 21)GGPRRHLGRRGH; (SEQ ID NO: 22) VSKRGHRRTAHE; (SEQ ID NO: 23) RGTRSGSTR;(SEQ ID NO: 24) RRRKKIQGRSKR; (SEQ ID NO: 25) RKSYGKYQGR;(SEQ ID NO: 26) KNGRYSISR; (SEQ ID NO: 27) RRRCGQKKK; (SEQ ID NO: 28)KQKIKHVVKLK; (SEQ ID NO: 29) KLKSQLVKRK; (SEQ ID NO: 30) RYPISRPRKR;(SEQ ID NO: 31) KVGKSPPVR; (SEQ ID NO: 32) KTFGKMKPR; (SEQ ID NO: 33)RIKWSRVSK;  and (SEQ ID NO: 34) KRTMRPTRR,

or (ii) an amino acid sequence having at least 90% sequence identity toan amino acid sequence of (i).
 17. The method of claim 14 wherein theglycan is selected from the group consisting of dextran, chondroitin,chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin,keratin, and keratan sulfate.
 18. The method of claim 14 wherein thesynthetic peptidoglycan is resistant to aggrecanase.
 19. The method ofclaim 14 wherein each synthetic peptide has a glycine-cysteine attachedto the C-terminus of the peptide.
 20. The method of claim 14 wherein thearthritis is selected from the group consisting of osteoarthritis andrheumatoid arthritis.
 21. The method of claim 14 wherein the dosage ofthe synthetic peptidoglycan is in a concentration ranging from about 0.1μM to about 10 μM. 22-24. (canceled)
 25. The synthetic peptidoglycan ofclaim 1, wherein the synthetic peptide comprises the amino acid sequenceGAHWQFNALTVRGG (SEQ ID NO: 2), or an amino acid sequence having at leastabout 90% sequence identity to GAHWQFNALTVRGG (SEQ ID NO: 2).
 26. Thesynthetic peptidoglycan of claim 1, wherein the synthetic peptidoglycancomprises from 2 to 20 of the synthetic peptides.
 27. The syntheticpeptidoglycan of claim 1, wherein the synthetic peptidoglycan comprisesfrom 5 to 15 of the synthetic peptides.
 28. The synthetic peptidoglycanof claim 1, wherein the synthetic peptides are covalently conjugated tothe glycan.
 29. The synthetic peptidoglycan of claim 28, wherein thesynthetic peptides are covalently conjugated to the glycan through alinker.
 30. The The synthetic peptidoglycan of claim 1, wherein thesynthetic peptides are conjugated to backbone of the glycan.